U.S. patent number 11,180,760 [Application Number 14/383,982] was granted by the patent office on 2021-11-23 for identification of molecular pathways and methods of use thereof for treating retinal neurodegeneration and other neurodegenerative disorders.
This patent grant is currently assigned to THE JOHNS HOPKINS UNIVERSITY. The grantee listed for this patent is THE JOHNS HOPKINS UNIVERSITY. Invention is credited to Derek S. Welsbie, Donald J. Zack.
United States Patent |
11,180,760 |
Zack , et al. |
November 23, 2021 |
Identification of molecular pathways and methods of use thereof for
treating retinal neurodegeneration and other neurodegenerative
disorders
Abstract
Drug targets, pathways, kits and methods for treating conditions
related to neurodegeneration or ocular disease, are disclosed.
Inventors: |
Zack; Donald J. (Baltimore,
MD), Welsbie; Derek S. (Lutherville-Timonium, MD) |
Applicant: |
Name |
City |
State |
Country |
Type |
THE JOHNS HOPKINS UNIVERSITY |
Baltimore |
MD |
US |
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Assignee: |
THE JOHNS HOPKINS UNIVERSITY
(Baltimore, MD)
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Family
ID: |
1000005950805 |
Appl.
No.: |
14/383,982 |
Filed: |
March 11, 2013 |
PCT
Filed: |
March 11, 2013 |
PCT No.: |
PCT/US2013/030203 |
371(c)(1),(2),(4) Date: |
September 09, 2014 |
PCT
Pub. No.: |
WO2013/134766 |
PCT
Pub. Date: |
September 12, 2013 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20150030572 A1 |
Jan 29, 2015 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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61609026 |
Mar 9, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N
15/1137 (20130101); C12Q 1/6883 (20130101); A61K
31/553 (20130101); C12Q 1/485 (20130101); A61K
31/713 (20130101); C12Q 2600/136 (20130101); C12N
2320/30 (20130101); C12Y 207/10 (20130101); C12N
2310/14 (20130101); G01N 2500/04 (20130101); C12Y
207/99 (20130101); C12Y 207/11 (20130101) |
Current International
Class: |
A61K
31/553 (20060101); C12N 15/113 (20100101); A61K
31/713 (20060101); C12Q 1/48 (20060101); C12Q
1/6883 (20180101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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2010017541 |
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Feb 2010 |
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WO |
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2011050192 |
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Apr 2011 |
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WO |
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2011119777 |
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Sep 2011 |
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WO |
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Other References
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self-destruction program promotes Wallerian degeneration, Nature
Neuroscience vol. 12 [ No. 4 [ Apr. 2009. cited by examiner .
Canola et al., Retinal Stem Cells Transplanted into Models of Late
Stages of Retinitis Pigmentosa Preferentially Adopt a Glial or a
Retinal Ganglion Cell Fate, Invest Ophthalmol Vis Sci.
2007;48:446-454) DOI:10.1167/iovs.06-0190. cited by examiner .
Itoh et al., ZPK/DLK, a Mitogen-Activated Protein Kinase Kinase
Kinase, Is a Critical Mediator of Programmed Cell Death of
Motoneurons, The Journal of Neuroscience, May 18, 2011
31(20):7223-7228 7223. cited by examiner .
Ballios et al., Biology and therapeutic potential of adult retinal
stem cells, Can J Ophthalmol 2010;45:342-51. cited by examiner
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Collins et al., Highwire Restrains Synaptic Growth by Attenuating a
MAP Kinase Signal, Neuron 51, 57-69, Jul. 6, 2006. cited by
examiner .
Nihalani et al., Identification of Structural and Functional
Domains in Mixed Lineage Kinase Dual Leucine Zipper-bearing Kinase
Required for Complex Formation and Stress-activated Protein Kinase
Activation, The Journal of Biological Chemistry, vol. 275, No. 10,
Issue of Mar. 10, pp. 7273-7279, 2000. cited by examiner .
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cDNA Encoding a New Member of Mixed Lineage Protein Kinase from
Human Brain, The Journal of Biological Chemistry, vol. 272, No. 45,
Issue of Nov. 7, pp. 28622-28629, 1997. cited by examiner .
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regulation by RNAi, Nature Biotechnology, Brief Communications, May
18, 2003 (Year: 2003). cited by examiner .
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competent gene silencing, Cancer Gene Therapy (2016) 23, 73-82,
(Year: 2016). cited by examiner .
Written Opinion dated Jun. 21, 2013 from PCT International
Application No. PCT/US2013/030203. cited by applicant .
Beirowski et al., "The WldS gene delays axonal but not somatic
degeneration in a rat glaucoma model." Eur J Neurosci. Sep. 2008;
28(6):1166-79. cited by applicant .
Chen et al., "Leucine Zipper-bearing Kinase promotes axon growth in
mammalian central nervous system neurons." Sci Rep. Aug. 11, 2016;
6:31482. cited by applicant .
Dickson et al., "POSH is an intracellular signal transducer for the
axon outgrowth inhibitor Nogo66." J Neurosci. Oct. 6, 2010;
30(40):13319-25. cited by applicant .
Ghosh et al., "DLK induces developmental neuronal degeneration via
selective regulation of proapoptotic JNK activity." J Cell Biol.
Sep. 5, 2011; 194(5):751-64. cited by applicant .
Ghosh-Roy et al., "Calcium and cyclic AMP promote axonal
regeneration in Caenorhabditis elegans and require DLK-1 kinase." J
Neurosci. Mar. 3, 2010; 30(9):3175-83. cited by applicant .
Hirai et al., "The c-Jun N-terminal kinase activator dual leucine
zipper kinase regulates axon growth and neuronal migration in the
developing cerebral cortex." J Neurosci. Nov. 15, 2006;
26(46):11992-2002. cited by applicant .
Libby et al., "Susceptibility to neurodegeneration in a glaucoma is
modified by Bax gene dosage." PLoS Genet. Jul. 2005; 1(1):17-26.
cited by applicant .
Sakuma et al., "Molecular cloning and functional expression of a
cDNA encoding a new member of mixed lineage protein kinase from
human brain." J Biol Chem. Nov. 7, 1997; 272(45):28622-9. cited by
applicant .
Yang et al., "Pathological axonal death through a MAPK cascade that
triggers a local energy deficit." Cell. Jan. 15,
2015;160(1-2):161-76. cited by applicant.
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Primary Examiner: Humphrey; Louise W
Assistant Examiner: Patury; Srikanth
Attorney, Agent or Firm: Casimir Jones, S.C. Isenbarger;
Thomas A.
Government Interests
FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
This invention was made with government support under 5R21EY019737
and 1K08EY022078 and awarded by the National Institutes of Health.
The government has certain rights in the invention.
Parent Case Text
RELATED APPLICATIONS
This application is a 35 U.S.C. .sctn. 371 National Stage Entry of
International Application No. PCT/US2013/030203 having an
international filing date of Mar. 11, 2013, which claims the
benefit of priority to U.S. Provisional Application No. 61/609,026,
filed Mar. 9, 2012, the entire disclosure of which are incorporated
herein in their entirety.
Claims
That which is claimed:
1. A method for inhibiting retinal ganglion cell injury or death,
the method comprising: contacting the retinal ganglion cell with a
first short interfering RNA (siRNA) that inhibits the expression or
activity of Mitogen-Activated Protein Kinase Kinase Kinase 12
(MAP3K12) and a second siRNA that inhibits the expression or
activity of Mitogen-Activated Protein Kinase Kinase Kinase 13
(MAP3K13).
2. The method of claim 1, wherein the retinal ganglion cell is
contacted with the first siRNA and second siRNA ex vivo or in
vivo.
3. The method of claim 2, wherein the method further comprises
grafting or implanting the retinal ganglion cell into a subject
after contacting the cell with the first siRNA and the second
siRNA.
4. The method of claim 3, wherein the retinal ganglion cell is in a
pharmaceutically acceptable carrier.
5. The method of claim 1, wherein inhibiting retinal ganglion cell
injury or death treats an ocular neurodegenerative disease.
6. The method of claim 5, wherein the ocular neurodegenerative
disease is selected from the group consisting of glaucoma, retinal
degeneration, and age-related macular degeneration.
Description
BACKGROUND
Neurodegenerative disorders afflict numerous patients throughout
the world and can be devastating to patients and caregivers. Such
disorders also can result in great financial burdens, with annual
costs currently exceeding several hundred billion dollars in the
United States alone. Current treatments for such disorders often
are inadequate. Further, many such disorders are age-related, and
thus their incidence is rapidly increasing as demographics trend
toward an aging population. One such disease, glaucoma, results in
damage to the optic nerve and is a major cause of vision loss and
blindness, especially in the elderly. Although various treatments
for glaucoma exist, many such treatments are of limited efficacy
and/or have significant side effects. Reduction of intraocular
pressure, generally through pharmacologic or surgical intervention,
is presently the mainstay of glaucoma therapy. Such therapies,
however, often are only partially effective and generally cannot
restore neuronal cell function once such function has been lost.
Other examples include retinal degeneration, such as retinitis
pigmentosa and the atrophic ("dry") form of age-related macular
degeneration (dAMD), which are types of retinal neurodegeneration
that can cause significant visual loss and blindness and currently
are essentially untreatable.
SUMMARY
The presently disclosed subject matter describes the identification
of pathways, drug targets, and biomarkers related to retinal
degenerative diseases including, but not limited to, glaucoma,
photoreceptor degeneration, such as retinitis pigmentosa and
age-related degeneration, and describes the modulation of these
targets as approaches for treating retinal and other
neurodegeneration.
In one aspect, the presently disclosed subject matter describes a
method for inhibiting or preventing retinal ganglion cell injury or
death, the method comprising contacting the retinal ganglion cell
with a small molecule that modulates protein kinase expression or
activity. Examples of the protein kinase include MAP3K12 (also
known as DLK), MAP3K13, MAP3K14, MAP2K7, MAP2K4, LYN, PLK3, PFKP
MARK3, MARK2, TAOK1, IKBKB, BRSK2, PBK, PRKCH, TESK1, Csnk1e,
Oxsr1, Tgfbr2, Mapk10, PFTK1, ERN2, AK2, HSPB8, FRAP1, DGUOK, ERN1,
STK32B, PIK3C2G, BCR, DYRK1A, DYRK1B, PNCK, EIF2AK1, PKD2L1, NRK,
Endothelin Receptor Type B, and TLK2. The modulation of protein
kinase activity or expression may be an inhibition or increase in
protein kinase activity or expression. In one aspect, the small
molecule is a short interfering or silencing RNA (siRNA). In a
further aspect, the siRNA targets MAP3K12, MAP3K13, MAP2K4, or
MAP2K7. In another aspect, the small molecule is selected from the
group consisting of CEP-1347 and CEP-11004.
In another aspect, the presently disclosed subject matter provides
a method for preventing or treating an ocular neurodegenerative
disease in a subject in need thereof, the method comprising
administering to the subject a therapeutically effective amount of
a small molecule which modulates protein kinase expression and/or
activity. Examples of the protein kinase include MAP3K12, MAP3K13,
MAP3K14, MAP2K7, MAP2K4, LYN, PLK3, PFKP, MARK3, MARK2, TAOK1,
IKBKB, BRSK2, PBK, PRKCH, TESK1, Csnk1e, Oxsr1, Tgfbr2, Mapk10,
PFTK1, ERN2, AK2, HSPB8, FRAP1, DGUOK, ERN1, STK32B, PIK3C2G, BCR,
DYRK1A, DYRK1B, PNCK, EIF2AK1, PKD2L1, NRK, Endothelin Receptor
Type B, and TLK2. In one aspect, the small molecule is a siRNA that
targets MAP3K12, MAP3K13, MAP2K4, or MAP2K7. In another aspect, the
small molecule is selected from the group consisting of CEP-1347
and CEP-11004.
In a further aspect, the presently disclosed subject matter
provides a method for identifying injury of a neuron or retinal
pigment epithelial (RPE) cell, the method comprising measuring the
amount of expression or activity of a target protein kinase in the
neuron or RPE cell; and determining if the amount of protein kinase
expression or activity in the neuron or RPE cell is greater than
the amount of protein kinase expression or activity in a control
neuron or RPE cell; wherein a determination that the amount of
protein kinase expression or activity is greater in the neuron or
RPE cell compared to the control neuron or RPE cell is indicative
of injury in the neuron. In a particular aspect, the target protein
kinase is selected from the group consisting of MAP3K12, MAP3K13,
MAP2K4, and MAP2K7. In another particular aspect, the neuron and
control neuron are retinal ganglion cells or photoreceptor
cells.
In a still further aspect, the presently disclosed subject matter
provides a method for identifying a gene encoding a protein kinase
associated with neuronal or retinal pigment epithelial (RPE) cell
injury or death. In one aspect, the method comprises: providing a
neuron or RPE cell; contacting the neuron or RPE cell with at least
one silencing RNA (siRNA) targeting a protein kinase in an amount
sufficient to inhibit or increase kinase activity of the protein
kinase; and determining whether the neuron or RPE cell survives;
wherein a determination that the neuron or RPE cell survives is an
indication that the gene encoding the protein kinase is associated
with neuronal or RPE cell injury or death. In a particular aspect,
the target protein kinase is selected from the group consisting of
MAP3K12, MAP3K13, MAP2K4, and MAP2K7. In another particular aspect,
the neuron is a retinal ganglion cell or a photoreceptor cell.
BRIEF DESCRIPTION OF THE FIGURES
Having thus described the presently disclosed subject matter in
general terms, reference will now be made to the accompanying
Figures, which are not necessarily drawn to scale, and wherein:
FIGS. 1A-1D show that broad-spectrum kinase inhibitors, sunitinib
and VX680, are neuroprotective to primary retinal ganglion cells
(RGCs) in vitro and in vivo: (A) primary RGCs plated in
neurotrophin-depleted media and stained with calcein AM at 72 hours
in the absence or presence of 1 .mu.M sunitinib; (B) survival of
primary RGCs in response to increasing doses of sunitinib and
VX680; (C and D) RGC survival in C57Bl/6 mice after injection
intravitreally with 1 .mu.L of drug-eluting microspheres containing
0.125, 0.25, or 0.5 mg/mL of sunitinib;
FIGS. 2A and 2B show knockdown in primary RGCs using
magnetofection-based siRNA delivery: (A) RGCs were transfected at
the time of isolation with 20-nM siGLO-red with (bottom) or without
(top) NeuroMag and stained for viability at 24 hours with calcein
AM; and (B) RGCs were transfected with increasing amounts of siRNA
against GAPDH (G) or a nontargeting control (C) and immunoblotted
for GAPDH or .beta.-tubulin expression at 48 hours;
FIGS. 3A-3F show the screening of a kinase-enriched siRNA library
in primary RGCs to identify a validated neuroprotective target: (A)
RGCs were plated in 96-well format and transfected with 2112 siRNAs
(20 nM) targeting 704 genes. Survival at 72 hours for any given
well was normalized to the mean of the 6 control wells (defined as
1.0); (B) shown are the top three siRNA candidates; (C)
confirmation screen using an independent set of siGenome or
On-Target plus siRNAs; (D) primary RGCs were transfected with
siRNAs against the various MAP kinases alone or in combination and
survival was measured; (E) RGCs transfected with siRNAs against
both MAP3K12/DLK and MAP3K13 were followed over time with calcein
AM staining; and (F) RGCs were transfected with 10 nM siRNA against
MAP3K12/DLK and MAP3K13 alone or in combination and then plated in
0, 12, 37, 111, 333, or 1000 nM sunitinib (left) or VX680 (right).
Survival at 72 hours is shown;
FIGS. 4A and 4B show the upregulation of MAP3K12/DLK in response to
retinal ganglion cell injury: (A) MAP3K12/DLK protein (top) and
mRNA (bottom) were measured by immunoblotting and quantitative
RT-PCR, respectively. GAPDH was used as loading control for both;
(B) C57Bl/6 mice were subjected to unilateral optic nerve crush,
the mice were sacrificed and the retinas were cryosectioned and
stained for DAPI (top) or MAP3K12/DLK (bottom). Shown is a
representative section demonstrating injury-induced upregulation of
MAP3K12/DLK;
FIG. 5 shows retinal ganglion cell survival after the cells were
transfected with MAP3K12/DLK siRNA;
FIG. 6A-6F demonstrate identification of MAP3K12/DLK as a mediator
of cell death in RGCs: (A) RGCs were transfected at the time of
immunopanning with a fluorescently-labeled siRNA (siGLO-Red,
Dharmacon) in the presence or absence of the magnetic nanoparticle,
NeuroMag. After 24 hours, RGCs were imaged for viability
(calcein-AM staining) and nuclear accumulation of siRNA; (B)
Histogram showing the normalized survival for control (black bars),
kinome library (light bars), MAP3K12/DLK (solid arrows) and MKK7
(dashed arrows) siRNAs. Oligonucleotides conferring survival more
than 3 SD from the nontargeting siRNAs (dashed line) were
considered neuroprotective (106 siRNA, 5.4%); (C) RGCs were
transfected with MAP3K12/DLK or a nontargeting control (NT) siRNA.
mRNA (left) and protein (right) levels were quantified at 24 hours
using RT-PCR and immunoblotting, respectively; (D) Survival of
immunopanned RGCs transfected with nontargeting (dashed) or
MAP3K12/DLK siRNA (solid); (E) Primary RGCs were transfected with
MAP3K12/DLK or nontargeting control (NT) siRNA. After the indicated
period of time (prior to cell death), cells were fixed and stained
for Brn3 expression; and (F) Patch-clamp recordings from RGCs
maintained with MAP3K12/DLK siRNA in response to depolarizing
current;
FIG. 7 shows efficient transfection of primary RGCs. RGCs were
reverse transfected with increasing doses of GAPDH or control small
interfering RNA oligonucleotide in the presence of a fixed amount
of NeuroMag and immunoblotted for GAPDH protein 24 h later;
FIGS. 8A-8B show secondary screening that confirmed the
neuroprotective activity of dual leucine zipper kinase and MKK7
small interfering RNA oligonucleotides (siRNAs): (A) RGCs were
immunopanned and transfected with an independent set of siRNAs not
used in the initial screen. Candidate genes were considered
confirmed if 75% of the secondary screening siRNA increased
survival more than three SDs above the control siRNAs (dashed
line); (B) RGCs were isolated from wild-type or Dlk.sup.fl/fl mice
and immediately transduced with adeno-GFP or adeno-Cre. After 72 h,
RGCs were imaged for viability by calcein-AM staining;
FIGS. 9A-9E show genetic deletion of MAP3K12/DLK protects RGCs from
axonal injury-induced cell death in vivo: (A) Dlk+/+ mice were
intravitreally injected with AAV2-Cre. Seven days after infection,
retinal flatmounts were stained for .beta.III tubulin and Cre; (B)
Three-month-old Dlk+/+ or Dlkfl/fl mice were intravitreally
injected with AAV2-Cre. Seven days later, eyes were subjected to
optic nerve crush. 4 days after injury, retinal flatmounts were
prepared and stained for MAP3K12/DLK; (C) Survival of RGCs 10 days
after optic nerve crush in Dlkfl/fl mice (n=7), Dlkfl/fl mice
injected with AAV2-Cre (n=8) or Dlk+/+ mice injected with AAV2-Cre
(n=9), normalized to uninjured controlmice (n=6). Representative
images shown to the right. Immunofluorescent staining of optic
nerves (D) and retinas (E) 24 hours after nerve crush in the mice
described in (C). *p<0.05, #p<0.005; Error bars show standard
deviation; and
FIGS. 10A-10C show MAP3K12/DLK protein is upregulated in RGCs in
response to injury: (A) Levels of MAP3K12/DLK protein (top) and
mRNA (bottom), normalized to GAPDH, after various times in culture;
(B) MAP3K12/DLK immunofluorescence of retinal sections 72 hours
after optic nerve transection in rats; and (C) Survival, measured
by CellTiter-Glo (CTG) luminescence, of immunopanned RGCs 48 hours
after transduction with adenovirus expressing wildtype (WT) or
kinase-dead (KD) MAP3K12/DLK. Western blot showing the upregulation
of MAP3K12/DLK protein and corresponding response of the JNK
pathway. *p<0.05; Error bars show standard deviation.
DETAILED DESCRIPTION
The presently disclosed subject matter now will be described more
fully hereinafter with reference to the accompanying Figures, in
which some, but not all embodiments of the presently disclosed
subject matter are shown. Like numbers refer to like elements
throughout. The presently disclosed subject matter may be embodied
in many different forms and should not be construed as limited to
the embodiments set forth herein; rather, these embodiments are
provided so that this disclosure will satisfy applicable legal
requirements. Indeed, many modifications and other embodiments of
the presently disclosed subject matter set forth herein will come
to mind to one skilled in the art to which the presently disclosed
subject matter pertains having the benefit of the teachings
presented in the foregoing descriptions and the associated Figures.
Therefore, it is to be understood that the presently disclosed
subject matter is not to be limited to the specific embodiments
disclosed and that modifications and other embodiments are intended
to be included within the scope of the appended claims.
The following abbreviations are used throughout the specification
and claims:
MAPK=mitogen-activated protein kinase spkl;
DLK=dual leucine zipper kinase;
LZK=leucine zipper-bearing kinase;
MAPKK=MAP2K, MAPK kinase;
MAPKKK=MAP3K, MAPKK kinase;
MKK=mitogen-activated protein kinase;
LYN=tyrosine-protein kinase encoded by the LYN gene;
PLK=polo-like kinase;
PFKP=phosphofructokinase, platelet;
MARK=microtubule affinity-regulating kinase;
TAOK=TAO kinase or serine/threonine protein kinase;
IKBKB=inhibitor of nuclear factor kappa-B kinase subunit beta;
BRSK=BR serine/threonine protein kinase;
PBK=lymphokine activated killer T cell originated protein
kinase;
PRKCH=protein kinase C eta type;
TESK1=dual specificity testis-specific protein kinase 1;
Csnk1e=casein kinase 1 isoform epsilon;
Oxsr1=serine/threonine-protein kinase OSR1;
Tgfbr2=transforming growth factor, beta receptor II;
PFTK=transketolase;
ERN2=Serine/threonine-protein kinase/endoribonuclease IRE2;
ERN1=Serine/threonine-protein kinase/endoribonuclease IRE1;
AK2=adenylate kinase;
HSPB8=heat shock protein beta-8;
FRAP1=serine/threonine-protein kinase mTOR;
DGUOK=deoxyguanosine kinase, mitochondrial isoform b;
STK32B=serine/threonine-protein kinase 32B;
PIK3C2G=phosphatidylinositol-4-phosphate 3-kinase C2
domain-containing subunit gamma;
DYRK1A=dual specificity tyrosine-phosphorylation-regulated kinase
1A;
BCR=breakpoint cluster region;
PNCK=calcium/calmodulin-dependent protein kinase type 1B or
pregnancy upregulated non-ubiquitously expressed CaM kinase;
EIF2AK1=Eukaryotic translation initiation factor 2-alpha kinase
1;
PKD2L1=Polycystic kidney disease 2-like 1 protein;
TLK2=serine/threonine-protein kinase tousled-like 2; and
MLK=mixed-lineage kinase.
I. Compounds, Compositions, and Methods for Treating
Neurodegenerative Disorders
Glaucoma, retinal degeneration (RD), and dAMD are a major cause of
visual loss and blindness in America and throughout the world. One
approach for treating glaucoma and other optic nerve diseases, as
well as other neurodegenerative diseases, disorders, or conditions
is through neuroprotective agents that promote the survival of
neurons or a portion thereof (e.g., the neuron cell body, an axon,
and/or a dendrite).
It previously has been shown that protein kinase inhibitors
identified through a high content screen of libraries of small
molecule compounds can promote the survival and/or neurite
outgrowth of retinal ganglion cells (RGCs), photoreceptor, and RPE
cells. They are active both in vitro and in vivo in animal models
of optic nerve injury, and also in a rat photoreceptor degeneration
model. See, for example, International PCT Patent Application
Publication Nos., WO2010/017541, to Zack et al., published Feb. 11,
2010, and WO2011/119777 to Zack et al., published Sep. 29, 2011,
each of which is incorporated by reference in their entirety.
RGCs are cells in the retina that die in glaucoma and whose loss
leads to vision loss. Accordingly, based on the activity of the
presently disclosed compounds on RGCs, the presently disclosed
compounds can be used for treating glaucoma and/or other optic
nerve diseases. Further, based on their activity on other neurons,
e.g., photoreceptors and hippocampal cell cultures, the presently
disclosed compounds can be used to treat other neurodegenerative
diseases in which there is a decreased function and/or loss of
neurons. In addition, based on their activity on RPE cells, as well
as photoreceptor cells, the presently disclosed compounds can be
used to treat dAMD.
A. Drug Targets and Pathways
In some embodiments, the presently disclosed subject matter
identifies drug targets and pathways for treating retinal and other
neurodegeneration: One approach to treating glaucoma, other optic
nerve diseases, retinal degeneration, dAMD, as well as other
neurodegeneration, is to develop "neuroprotective" agents that
promote the survival of neurons. In some embodiments, the presently
disclosed subject matter provides a screen assay, using RGC neurons
damaged in glaucoma to identify novel drug targets. In further
embodiments, the presently disclosed screening assay has been used
to survey a portion of the mouse genome and, in doing so, has led
to the identification of several novel targets that are involved in
neuronal cell death (such that the cells survive when the targets
are inhibited). One of these targets is only expressed following
axonal damage and may represent a biomarker of neuronal injury in
glaucoma and other neurodegeneration.
Accordingly, the presently disclosed subject matter includes: (a)
design of a method to transfect retinal ganglion cell and screen
for new neuroprotective drug targets; (b) identification of a novel
neuroprotective drug target; (c) identification of a novel
biomarker of neuronal injury (all current diagnostic tests for
glaucoma measure anatomic and functional changes from retinal
ganglion cell death); and (d) identification of the relevant target
of neuroprotective protein kinase inhibitors.
Particular features of the presently disclosed subject matter
include: (a) a high-throughput genetic screen (and the transfection
method) to identify genes involved in the death and survival of
primary neurons; (b) genes identified as being involved in retinal
ganglion cell death. Further, for the first time, the targeting of
those genes by siRNA and small molecule inhibitors to treat retinal
ganglion cell degeneration (like glaucoma) is described; and (c)
identification of a gene that is selectively expressed in injured
neurons.
B. Methods of Treatment
In some embodiments, the presently disclosed subject matter
identifies molecular pathways involved in the mechanism by which
retinal ganglion cells (RGCs) die in glaucoma, and the same or
similar mechanisms also may represent the pathways by which
photoreceptor cells die in the retinal degeneration (RD) and in
dAMD, which also includes death of RPE cells. The pathways were
identified by a whole kinome screen in which short interfering or
silencing RNAs (siRNAs) were used to knockdown separately each of
the kinases in cultured RGCs, and the effect of such knockdown on
RGC survival was assayed.
In other aspects, the presently disclosed subject matter provides a
method for treating or preventing a neurodegenerative disease,
disorder, or condition in a subject in need thereof, the method
comprising administering to the subject a therapeutically effective
amount of a compound, such as a small molecule protein kinase
inhibitor, or a pharmaceutically acceptable salt thereof, that
inhibits one of the specific identified pathways or kinases,
thereby treating or preventing the neurodegenerative disease,
disorder, or condition; or use of a genomic reagent, such as a
siRNA, or a modified siRNA, that directly or indirectly modifies
the expression of the identified drug target, its biochemical
activity, or otherwise modifies the activity of the identified
pathway. In particular embodiments, the neurodegenerative disease,
disorder, or condition is an ocular-related neurodegeneration, such
as glaucoma, RD, or dAMD.
In one embodiment, the presently disclosed subject matter describes
a method for inhibiting or preventing retinal ganglion cell injury
or death, the method comprising contacting the retinal ganglion
cell with a small molecule that modulates protein kinase expression
or activity. Examples of the protein kinase include MAP3K12,
MAP3K13, MAP3K14, MAP2K7, MAP2K4, LYN, PLK3, PFKP MARK3, MARK2,
TAOK1, IKBKB, BRSK2, PBK, PRKCH, TESK1, Csnk1e, Oxsr1, Tgfbr2,
Mapk10, PFTK1, ERN2, AK2, HSPB8, FRAP1, DGUOK, ERN1, STK32B,
PIK3C2G, BCR, DYRK1A, DYRK1B, PNCK, EIF2AK1, PKD2L1, NRK,
Endothelin Receptor Type B, and TLK2. The modulation of protein
kinase activity or expression may be an inhibition or increase in
protein kinase activity or expression. In one embodiment, the small
molecule is a short interfering or silencing RNA (siRNA). In a
further embodiment, the siRNA targets are selected from the group
consisting of MAP3K12, MAP3K13, MAP2K4, and MAP2K7.
The contacting of the cell with a small molecule can be performed
ex vivo or in vivo. It may further comprise grafting or implanting
the cell into a subject after contacting the cell with a small
molecule. The grafting or implanting the cell into a subject can
occur in a pharmaceutically acceptable carrier. The inhibiting or
preventing retinal ganglion cell injury or death may treat or
prevent an ocular neurodegenerative disease, such as glaucoma,
retinal degeneration, or age-related macular degeneration. The
modulation of protein kinase expression or activity may be an
inhibition of expression or activity or an increase in expression
or activity. The small molecule may be a short interfering RNA
(siRNA), such as a siRNA targeting MAP3K12, MAP3K13, MAP2K4, or
MAP2K7. Thus, the presently disclosed methods may involve
administering a small molecule, such as a siRNA, to a subject.
Administering to a subject may occur via a pharmaceutically
acceptable carrier.
In some embodiments, the ocular neurodegenerative disease is
selected from the group consisting of glaucoma, retinal
degeneration, and age-related macular degeneration.
In some embodiments, the presently disclosed subject matter
provides a compound that promotes neuroprotection by inhibiting any
of the molecules, activities, or pathways provided herein in Table
1. A specific example is the use of a small molecule to inhibit the
protein kinase MAP3K12 (also known as DLK) and other members of the
mixed-lineage kinase family to achieve neuroprotective treatment of
glaucoma, RD, dAMD, and other forms of ocular neurodegeneration, as
well as forms of CNS neurodegeneration. A more specific example is
the use of CEP-1347, CEP-11004, and other MLK inhibitors to promote
retinal neuronal health and survival.
TABLE-US-00001 TABLE 1 Drug/Pathway Targets MAP3K12/DLK (GenBank
Accession No. NM_006301) MAP3K13 (GenBank Accession No. NM_004721)
MAP3K14 (isoform 1) (RefSeq Accession No. NP_001180440) MAP2K7
(GenBank Accession No. AAH38295) MAP2K4 (GenBank Accession No.
CAG38801) LYN (RefSeq Accession No. NP_002341) PLK3 (RefSeq
Accession No. NP_004064) PFKP (GenBank Accession No. AAH02536)
MARK3 (isoform a) (RefSeq Accession No. NP_001122390) MARK2
(isoform a) (RefSeq Accession No. NP_059672) TAOK1 (GenBank
Accession No. AA144068) IKBKB (isoform 1) (RefSeq Accession No.
NP_001547) BRSK2 (isoform 1) (RefSeq Accession No. NP_001243558)
PBK (RefSeq Accession No. NP_060962) PRKCH (RefSeq Accession No.
NP_006246) TESK1 (RefSeq Accession No. NP_006276) Csnk1e (RefSeq
Accession No. NP_689407) Oxsr1 (RefSeq Accession No. NP_005100)
Tgfbr2 (GenBank Accession No. ABG65632) Mapk10 (GenBank Accession
No. AAH51731) PFTK1 (GenBank Accession No. AA136477) ERN2 (RefSeq
Accession No. NP_150296) AK2 (mitochondrial (RefSeq Accession No.
NP_001616) isoform a) HSPB8 (RefSeq Accession No. NP_055180) FRAP1
(RefSeq Accession No. NP_004949) DGUOK (mitochondrial (RefSeq
Accession No. NP_550440) isoform b) ERN1 (RefSeq Accession No.
NP_001424) STK32B (RefSeq Accession No. NP_060871) PIK3C2G (RefSeq
Accession No. NP_004561) BCR (isoform 1) (RefSeq Accession No.
NP_004318) DYRK1A (isoform 1) (RefSeq Accession No. NP_001387) PNCK
(GenBank Accession No. AAH64422) EIF2AK1 (isoform a) (RefSeq
Accession No. NP_055228) PKD2L1 (isoform 1) (RefSeq Accession No.
NP_057196) TLK2 (isoform A) (RefSeq Accession No. NP_006843)
Accordingly, in some embodiments, the MLK inhibitor is a compound
of Formula (I):
##STR00001## wherein:
R.sub.1 is selected from the group consisting of H, halogen, alkyl,
--OH, --NHCONHC.sub.6H.sub.5, CH.sub.2SOC.sub.2H.sub.5,
--NHCONHC.sub.2H.sub.5, --CH.sub.2SC.sub.2H.sub.5,
--CH.sub.2SC.sub.2H.sub.5, --NHCONHC.sub.2H.sub.5,
--R.sub.3R.sub.4, wherein R.sub.3 and R.sub.4 are each H or alkyl,
--CH.sub.2OCONHC.sub.2H.sub.5, --NHCO.sub.2CH.sub.3,
--CH.sub.2OC.sub.2H.sub.5, --CH.sub.2N(CH.sub.3).sub.2,
--CH.sub.2SO.sub.2C.sub.2H.sub.5, --CH.sub.2S--O.sub.5H.sub.4N,
--CH.sub.2SC.sub.2H.sub.5, --CH_NNH--O.sub.5N.sub.2H.sub.5,
--CH.sub.2S--C.sub.4N.sub.2H.sub.3,
--CH.sub.2S(O)--C.sub.4N.sub.2H.sub.3,
--CH.sub.2S(O)C.sub.5H.sub.4, --CH.sub.2SC.sub.2H.sub.5,
O-n-propyl, --CH.sub.2SCH.sub.2CH.sub.2N(CH.sub.3).sub.2,
--CH.sub.2S-benzimidazole, --CH.sub.2SCH.sub.2-furan,
--CH.dbd.N-pyrrolidine, --CH.dbd.NNH-pyridine,
--CH.sub.2S(CH.sub.2).sub.2NH.sub.2, --CH.sub.2-1,2,4-triazole,
--CH.dbd.NNH--C(.dbd.NH)NH.sub.2, --CH--N-1,2,4-triazole,
--CH.dbd.N-mopholine, --CHN--N(CH.sub.3).sub.2,
--CH.dbd.N-1-methylpiperazine,
--CHCH.sub.2S(CH.sub.2).sub.2NH-n-C.sub.4H.sub.9, and
--CH.sub.2S--(CH.sub.2).sub.2N(CH.sub.3).sub.2;
R.sub.2 is selected from the group consisting of H, halogen,
--NHCONHC.sub.2H.sub.5, --CH.sub.2SC.sub.2H.sub.2, --CH.sub.2OH,
--NH.sub.2, --CH.sub.2S(CH.sub.2).sub.2--N(CH.sub.3).sub.2;
X is selected from the group consisting of H, --CH.sub.2N.sub.3,
--CO.sub.2CH.sub.3, --CH.sub.2OH, --CONHC.sub.2H.sub.5,
--CH.dbd.NNH--C.sub.3N.sub.2H.sub.5, --CH.sub.2NH-Gly,
--CON(CH.sub.3).sub.2, --CH.sub.2NHCO.sub.2, --CONH.sub.2,
--CONHCC.sub.3H.sub.7, --CH.sub.2NH-Ser, --CH.sub.2SOCH.sub.3,
--CH.dbd.NOH, --C--O-morpholine, --CH.sub.2NH-Pro,
--CH.dbd.NNHC(.dbd.NH)NH.sub.2, --CONH(CH.sub.2)OH,
--CO.sub.2CH.sub.3, --CH.dbd.NNHCONH.sub.2, --CH.sub.2OCOCH.sub.3,
--CONHC.sub.6H.sub.5, --CH.sub.2SO-pyridine,
--CH.sub.2NHCO.sub.2C.sub.6H.sub.5, --CH.sub.2OH,
--CONHC.sub.6H.sub.5, --CONHCH.sub.2CH.sub.2OH,
CH.sub.2NHCO.sub.2CH.sub.3, --CONH.sub.2,
--CH.sub.2SC.sub.6H.sub.5, --CH.sub.2S-pyridine,
--CH.sub.2SOC.sub.6H.sub.5, --CO.sub.2-n-hexyl, --CH.sub.2NH.sub.2,
and --CONHCH.sub.3;
R is selected from the group consisting of --OH and -methoxyl;
Z.sub.1 and Z.sub.2 are selected from the group consisting H and O;
and
pharmaceutically acceptable salts thereof.
In particular embodiments, the MLK inhibitor is CEP-1347, which has
the following structure:
##STR00002##
In other embodiments, the MLK inhibitor is CEP-11004, which has the
following structure:
##STR00003##
In particular embodiments, the protein kinase inhibitor is a
compound disclosed in U.S. Pat. No. 5,621,100 or International PCT
Patent Application Publication No. WO9402488, each of which is
incorporated herein by reference in its entirety.
In other embodiments, the presently disclosed subject matter
provides a compound that promotes neuroprotection by stimulating
any of the molecules, activities, or pathways provided herein in
Table 1.
In further embodiments, the presently disclosed subject matter
provides a siRNA, modified siRNA, shRNA, modified shRNA, or other
molecule or method that promotes neuroprotection by decreasing
expression or activity of any of the molecules or pathways provided
herein in Table 1.
In yet further embodiments, the presently disclosed subject matter
provides a siRNA, modified siRNA, shRNA, modified shRNA, or other
molecule or method that promotes neuroprotection by increasing
expression or activity of any of the molecules or pathways provided
herein in Table 1.
A variety of means are available for altering a gene to effect
expression. In a particular embodiment the expression of a gene
encoding a protein kinase is reduced by contacting the gene, or an
mRNA transcribed from the gene, with a compound comprising a
polynucleotide selected from the group consisting of an antisense
oligonucleotide, a ribozyme, a small interfering RNA (siRNA), and a
short hairpin RNA (shRNA). In a particularly preferred embodiment
the compound comprises a nucleotide sequence complementary to a
nucleotide sequence comprising the polynucleotide sequence of
MAP3K12, MAP3K13, MAP2K4, or MAP2K7.
The term "polynucleotide" means a polynucleic acid, in single or
double stranded form, and in the sense or antisense orientation,
complementary polynucleic acids that hybridize to a particular
polynucleic acid under stringent conditions, and polynucleotides
that are homologous in at least about 60 percent of its base pairs,
and more preferably 70 percent of its base pairs are in common,
most preferably 90 percent, and in a special embodiment 100 percent
of its base pairs. The polynucleotides include polyribonucleic
acids, polydeoxyribonucleic acids, and synthetic analogues thereof.
The polynucleotides are described by sequences that vary in length,
that range from about 10 to about 5000 bases, preferably about 100
to about 4000 bases, more preferably about 250 to about 2500 bases.
A preferred polynucleotide embodiment comprises from about 10 to
about 30 bases in length. A particular embodiment of polynucleotide
is the polyribonucleotide of from about 10 to about 22 nucleotides,
more commonly described as small interfering RNAs (siRNAs). Another
special embodiment are nucleic acids with modified backcartilages
such as peptide nucleic acid (PNA), polysiloxane, and
2'-O-(2-methoxy)ethylphosphorothioate, or including non-naturally
occurring nucleic acid residues, or one or more nucleic acid
substituents, such as methyl-, thio-, sulphate, benzoyl-, phenyl-,
amino-, propyl-, chloro-, and methanocarbanucleosides, or a
reporter molecule to facilitate its detection.
The term "antisense nucleic acid" refers to an oligonucleotide that
has a nucleotide sequence that interacts through base pairing with
a specific complementary nucleic acid sequence involved in the
expression of the target such that the expression of the gene is
reduced. Preferably, the specific nucleic acid sequence involved in
the expression of the gene is a genomic DNA molecule or mRNA
molecule that encodes (a part of) the gene. This genomic DNA
molecule can comprise regulatory regions of the gene, or the coding
sequence for the mature gene.
The term "complementary to a nucleotide sequence" in the context of
antisense oligonucleotides and methods should be understood as
sufficiently complementary to such a sequence as to allow
hybridization to that sequence in a cell, i.e., under physiological
conditions.
The term "hybridization" means any process by which a strand of
nucleic acid binds with a complementary strand through base
pairing. The term "hybridization complex" refers to a complex
formed between two nucleic acid sequences by virtue of the
formation of hydrogen bonds between complementary bases. A
hybridization complex may be formed in solution or formed between
one nucleic acid sequence present in solution and another nucleic
acid sequence immobilized on a solid support (e.g., paper,
membranes, filters, chips, pins or glass slides, or any other
appropriate protein kinase to which cells or their nucleic acids
have been fixed). The term "stringent conditions" refers to
conditions that permit hybridization between polynucleotides and
the claimed polynucleotides. Stringent conditions can be defined by
salt concentration, the concentration of organic solvent, e.g.,
formamide, temperature, and other conditions well known in the art.
In particular, reducing the concentration of salt, increasing the
concentration of formamide, or raising the hybridization
temperature can increase stringency.
The down regulation of gene expression using antisense nucleic
acids can be achieved at the translational or transcriptional level
using an expression-inhibitory agent. Antisense nucleic acids of
the invention are preferably nucleic acid fragments capable of
specifically hybridizing with all or part of a nucleic acid
encoding a protein kinase or the corresponding messenger gene or
mRNA. In addition, antisense nucleic acids may be designed which
decrease expression of the nucleic acid sequence capable of
encoding a protein kinase by inhibiting splicing of its primary
transcript. Any length of antisense sequence is suitable for
practice of the invention so long as it is capable of
down-regulating or blocking expression of a nucleic acid coding for
a protein kinase. Preferably, the antisense sequence is at least
about 17 nucleotides in length. The preparation and use of
antisense nucleic acids, DNA encoding antisense RNAs and the use of
oligo and genetic antisense is known in the art.
The term "expression inhibitory agent" means a polynucleotide
designed to interfere selectively with the transcription,
translation and/or expression of a specific polypeptide or protein
normally expressed within a cell. More particularly, "expression
inhibitory agent" comprises a DNA or RNA molecule that contains a
nucleotide sequence identical to or complementary to at least about
17 sequential nucleotides within the polyribonucleotide sequence
coding for a specific polypeptide or protein. Exemplary expression
inhibitory molecules include ribozymes, double stranded siRNA
molecules, self-complementary single-stranded siRNA molecules,
genetic antisense constructs, and synthetic RNA antisense molecules
with modified stabilized backbones.
One embodiment of expression-inhibitory agent is a nucleic acid
that is antisense to a nucleotide sequence comprising the
polynucleotide sequence of MAP3K12, MAP3K13, MAP2K4, or MAP2K7. For
example, an antisense nucleic acid (e.g. DNA) may be introduced
into cells in vitro, or administered to a subject in vivo, as gene
therapy to inhibit cellular expression of nucleotide sequence
comprising the polynucleotide sequence of MAP3K12, MAP3K13, MAP2K4,
or MAP2K7. Antisense oligonucleotides preferably comprise a
sequence containing from about 17 to about 100 nucleotides and more
preferably the antisense oligonucleotides comprise from about 18 to
about 30 nucleotides. Antisense nucleic acids may be prepared from
about 10 to about 30 contiguous nucleotides complementary to a
nucleotide sequence comprising the polynucleotide sequence of
MAP3K12, MAP3K13, MAP2K4, or MAP2K7.
The antisense nucleic acids are preferably oligonucleotides and may
consist entirely of deoxyribo-nucleotides, modified
deoxyribonucleotides, or some combination of both. The antisense
nucleic acids can be synthetic oligonucleotides. The
oligonucleotides may be chemically modified, if desired, to improve
stability and/or selectivity. Since oligonucleotides are
susceptible to degradation by intracellular nucleases, the
modifications can include, for example, the use of a sulfur group
to replace the free oxygen of the phosphodiester bond. This
modification is called a phosphorothioate linkage. Phosphorothioate
antisense oligonucleotides are water soluble, polyanionic, and
resistant to endogenous nucleases. In addition, when a
phosphorothioate antisense oligonucleotide hybridizes to its mRNA
target, the RN.sub.2O.sub.2-315NA duplex activates the endogenous
enzyme ribonuclease (RNase) H, which cleaves the mRNA component of
the hybrid molecule.
In addition, antisense oligonucleotides with phosphoramidite and
polyamide (peptide) linkages can be synthesized. These molecules
should be very resistant to nuclease degradation. Furthermore,
chemical groups can be added to the 2' carbon of the sugar moiety
and the 5 carbon (C-5) of pyrimidines to enhance stability and
facilitate the binding of the antisense oligonucleotide to its
target site. Modifications may include 2'-deoxy, O-pentoxy,
O-propoxy, O-methoxy, fluoro, methoxyethoxy phosphorothioates,
modified bases, as well as other modifications known to those of
skill in the art.
Another type of expression-inhibitory agent that reduces the levels
of mRNA is the ribozyme. Ribozymes are catalytic RNA molecules (RNA
enzymes) that have separate catalytic and substrate binding
domains. The substrate binding sequence combines by nucleotide
complementarity and, possibly, non-hydrogen bond interactions with
its mRNA sequence. The catalytic portion cleaves the mRNA at a
specific site. The substrate domain of a ribozyme can be engineered
to direct it to a specified mRNA sequence. The ribozyme recognizes
and then binds protein kinase mRNA through complementary base
pairing. Once it is bound to the correct protein kinase mRNA site,
the ribozyme acts enzymatically to cut the protein kinase mRNA.
Cleavage of the mRNA by a ribozyme destroys its ability to direct
synthesis of the corresponding polypeptide. Once the ribozyme has
cleaved its protein kinase mRNA sequence, it is released and can
repeatedly bind and cleave at other mRNAs. Ribozyme forms include a
hammerhead motif, a hairpin motif, a hepatitis delta virus, group I
intron or RNaseP RNA (in association with an RNA guide sequence)
motif or Neurospora VS RNA motif Ribozymes possessing a hammerhead
or hairpin structure are readily prepared since these catalytic RNA
molecules can be expressed within cells from eukaryotic promoters
(Chen et al. Nucleic Acids Res. 20:4581-9, 1985). A ribozyme of the
presently disclosed subject matter can be expressed in eukaryotic
cells from the appropriate DNA vector.
If desired, the activity of the ribozyme may be augmented by its
release from the primary transcript by a second ribozyme (Ventura
et al. Nucleic Acids Res. 21:3249-55, 1983).
The term "vectors" relates to plasmids as well as to viral vectors,
such as recombinant viruses, or the nucleic acid encoding the
recombinant virus.
Ribozymes may be chemically synthesized by combining an
oligodeoxyribonucleotide with a ribozyme catalytic domain (20
nucleotides) flanked by sequences that hybridize to the protein
kinase mRNA after transcription. The oligodeoxyribonucleotide is
amplified by using the substrate binding sequences as primers. The
amplification product is cloned into a eukaryotic expression
vector.
Ribozymes are expressed from transcription units inserted into DNA,
RNA, or viral vectors. Transcription of the ribozyme sequences are
driven from a promoter for eukaryotic RNA polymerase I (pol (I),
RNA polymerase II (pol II), or RNA polymerase III (pol III).
Transcripts from pol II or pol III promoters will be expressed at
high levels in all cells; the levels of a given pol II promoter in
a given cell type will depend on nearby gene regulatory sequences.
Prokaryotic RNA polymerase promoters are also used, providing that
the prokaryotic RNA polymerase enzyme is expressed in the
appropriate cells (Gao and Huang, Nucleic Acids Res. 21:2867-72,
1993). It has been demonstrated that ribozymes expressed from these
promoters can function in mammalian cells (Kashani-Sabet et al.,
Antisense Res. Dev. 2:3-15, 1992).
A particularly preferred inhibitory agent is a small interfering
RNA (siRNA). siRNA, preferably short hairpin RNA (shRNA), mediate
the post-transcriptional process of gene silencing by double
stranded RNA (dsRNA) that is homologous in sequence to the silenced
RNA. siRNA according to the presently disclosed subject matter
comprises a sense strand of 17-25 nucleotides complementary or
homologous to a contiguous 17-25 nucleotide sequence of the
polynucleotide sequence of MAP3K12 (GenBank Accession No, NM
006301) or MAP3K13 (Genbank Accession Number NM 004721), and an
antisense strand of 17-23 nucleotides complementary to the sense
strand. The most preferred siRNA comprises sense and anti-sense
strands that are 100 percent complementary to each other and the
protein kinase polynucleotide sequence. Preferably the siRNA
further comprises a loop region linking the sense and the antisense
strand. A self-complementing single stranded siRNA molecule
polynucleotide according to the presently disclosed subject matter
comprises a sense portion and an antisense portion connected by a
loop region linker. The loop can be any length but is preferably
4-30 nucleotides long. Self-complementary single stranded siRNAs
form hairpin loops and are more stable than ordinary dsRNA. In
addition, they are more easily produced from vectors.
Analogous to antisense RNA, the siRNA can be modified to confirm
resistance to nucleolytic degradation, or to enhance activity, or
to enhance cellular distribution, or to enhance cellular uptake,
such modifications may consist of modified internucleoside
linkages, modified nucleic acid bases, modified sugars and/or
chemical linkage the siRNA to one or more moieties or
conjugates.
The presently disclosed subject matter also relates to compositions
and methods comprising a DNA expression vector capable of
expressing a polynucleotide capable of increasing resistance to
cell damage and is described hereinabove as an expression
inhibition agent.
In another embodiment, the presently disclosed compositions and
methods relate to the down-regulation or blocking of the expression
of a protein kinase by the induced expression of a polynucleotide
encoding an intracellular binding protein that is capable of
selectively interacting with the protein kinase polypeptide. An
intracellular binding protein includes any protein capable of
selectively interacting, or binding, with the polypeptide in the
cell in which it is expressed and neutralizing the function of the
polypeptide. Preferably, the intracellular binding protein is a
neutralizing antibody or a fragment of a neutralizing antibody
having binding affinity to an epitope of the protein kinase. More
preferably, the intracellular binding protein is a single chain
antibody.
The term "binding affinity" is a property that describes how
strongly two or more compounds associate with each other in a
non-covalent relationship. Binding affinities can be characterized
qualitatively, (such as "strong", "weak", "high", or "low") or
quantitatively (such as measuring the KO.
In a particular embodiment, the composition comprises an
expression-inhibiting agent selected from the group consisting of
antisense RNA, antisense oligodeoxynucleotide (ODN), and a small
interfering RNA (siRNA) that is sufficiently homologous to a
portion of the polynucleotide encoding a target protein kinase such
that the siRNA interferes with the translation of the protein
kinase polyribonucleotide to the protein kinase polypeptide.
The polynucleotide expressing the expression-inhibiting agent is
preferably included within a vector. The polynucleic acid is
operably linked to signals enabling expression of the nucleic acid
sequence and is introduced into a cell utilizing, preferably,
recombinant vector constructs, which will express the antisense
nucleic acid once the vector is introduced into the cell. A variety
of viral-based systems are available, including adenoviral,
retroviral, adeno-associated viral, lentiviral, herpes simplex
viral or a sendaviral vector systems, and all may be used to
introduce and express polynucleotide sequence for the
expression-inhibiting agents in protein kinase-expressing
cells.
The term "operably linked" or "operably inserted" means that the
regulatory sequences necessary for expression of the coding
sequence are placed in a nucleic acid molecule in the appropriate
positions relative to the coding sequence so as to enable
expression of the coding sequence. This same definition is
sometimes applied to the arrangement other transcription control
elements (e.g. enhancers) in an expression vector. Transcriptional
and translational control sequences are DNA expression regulatory
sequences, such as promoters, enhancers, polyadenylation signals,
terminators, and the like, that provide for the expression of a
coding sequence in a host cell.
The terms "promoter", "promoter region" or "promoter sequence"
refer generally to transcriptional regulatory regions of a gene,
which may be found at the 5' or 3' side of the coding region, or
within the coding region, or within introns. Promoters that may be
used in the expression vectors of the presently disclosed subject
matter include both constitutive promoters and regulated
(inducible) promoters.
Typically, a promoter is a DNA regulatory region capable of binding
RNA polymerase in a cell and initiating transcription of a
downstream (3' direction) coding sequence. The typical 5' promoter
sequence is bounded at its 3' terminus by the transcription
initiation site and extends upstream (5' direction) to include the
minimum number of bases or elements necessary to initiate
transcription at levels detectable above background. Within the
promoter sequence is a transcription initiation site (conveniently
defined by mapping with nuclease S1), as well as protein binding
domains (consensus sequences) responsible for the binding of RNA
polymerase.
In a particular embodiment, the viral element is derived from an
adenovirus. Other embodiments of the presently disclosed subject
matter use retroviral vector systems which can be constructed from
different types of retrovirus, such as, MoMuLV ("murine Moloney
leukemia virus" MSV ("murine Moloney sarcoma virus"), HaSV ("Harvey
sarcoma virus"); SNV ("spleen necrosis virus"); RSV ("Rous sarcoma
virus") and Friend virus. Lentiviral vector systems may also be
used in the practice of the presently disclosed subject matter. In
other embodiments of the presently disclosed subject matter,
adeno-associated viruses ("AAV") may be utilized.
Preferably, the viral vectors used in the methods of the presently
disclosed subject matter are replication defective. Such
replication defective vectors will usually pack at least one region
that is necessary for the replication of the virus in the infected
cell. These regions can either be eliminated (in whole or in part),
or be rendered non-functional by any technique known to a person
skilled in the art. These techniques include the total removal,
substitution, partial deletion or addition of one or more bases to
an essential (for replication) region. Such techniques may be
performed in vitro (on the isolated DNA) or in situ, using the
techniques of genetic manipulation or by treatment with mutagenic
agents. Preferably, the replication defective virus retains the
sequences of its genome, which are necessary for encapsidating, the
viral particles.
In the vector construction, the polynucleotide agents of the
presently disclosed subject matter may be linked to one or more
regulatory regions. Selection of the appropriate regulatory region
or regions is a routine matter, within the level of ordinary skill
in the art. Regulatory regions include promoters, and may include
enhancers, suppressors, and the like.
Additional vector systems include the non-viral systems that
facilitate introduction of polynucleotide agents into a patient.
For example, a DNA vector encoding a desired sequence can be
introduced in vivo by lipofection. Synthetic cationic lipids
designed to limit the difficulties encountered with
liposome-mediated transfection can be used to prepare liposomes for
in vivo transfection of a gene encoding a marker (Feigner et. al.,
Proc. Natl. Acad. Sci. USA 84:7413-7, 1987); see Mackey et al.,
Proc. Natl. Acad. Sci. USA 85:8027-31, 1988; Ulmer et al., Science
259:1745-8, 1993). The use of cationic lipids may promote
encapsulation of negatively charged nucleic acids, and also promote
fusion with negatively charged cell membranes (Feigner and Ringold,
Nature 337:387-8, 1989). Particularly useful lipid compounds and
compositions for transfer of nucleic acids are described in
International PCT Patent Publications WO 95/18863 and WO 96/17823,
and in U.S. Pat. No. 5,459,127. The use of lipofection to introduce
exogenous genes into the specific organs in vivo has certain
practical advantages and directing transfection to particular cell
types would be particularly advantageous in a tissue with cellular
heterogeneity, for example, pancreas, liver, kidney, and the brain.
Lipids may be chemically coupled to other molecules for the purpose
of targeting. Targeted peptides, e.g., hormones or
neurotransmitters, and proteins for example, antibodies, or
non-peptide molecules could be coupled to liposomes chemically.
Other molecules are also useful for facilitating transfection of a
nucleic acid in vivo, for example, a cationic oligopeptide (e.g.,
International PCT Patent Publication WO 95/21931), peptides derived
from DNA binding proteins (e.g., International PCT Patent
Publication WO 96/25508), or a cationic polymer (e.g.,
International PCT Patent Publication WO 95/21931).
It is also possible to introduce a DNA vector in vivo as a naked
DNA plasmid (see U.S. Pat. Nos. 5,693,622, 5,589,466 and
5,580,859). Naked DNA vectors for therapeutic purposes can be
introduced into the desired host cells by methods known in the art,
e.g., transfection, electroporation, microinjection, transduction,
cell fusion, DEAE dextran, calcium phosphate precipitation, use of
a gene gun, or use of a DNA vector transporter (see, e.g., Wilson
et al., J. Biol. Chem. 267:963-7, 1992; Wu and Wu, J. Biol. Chem.
263:14621-4, 1988; Hartmut, et al. Canadian Patent Application No.
2,012,311, filed Mar. 15, 1990; Williams et al, Proc. Natl. Acad.
Sci. USA 88:2726-30, 1991). Receptor-mediated DNA delivery
approaches can also be used (Curiel, et al., Hum. Gene Ther.
3:147-54, 1992; Wu and Wu, J. Biol. Chem. 262:4429-32, 1987).
In particular embodiments, the neurodegenerative disease, disorder,
or condition is an ocular-related neurodegeneration. In more
particular embodiments, the ocular-related neurodegeneration is
selected from the group consisting of glaucoma, lattice dystrophy,
retinitis pigmentosa, age-related macular degeneration (AMD),
photoreceptor degeneration associated with wet or dry AMD, other
retinal degeneration, optic nerve drusen, optic neuropathy, and
optic neuritis, such as optic neuritis resulting from multiple
sclerosis.
In yet more particular embodiments, the glaucoma is selected from
the group consisting of primary glaucoma, low-tension glaucoma,
primary angle-closure glaucoma, acute angle-closure glaucoma,
chronic angle-closure glaucoma, intermittent angle-closure
glaucoma, chronic open-angle closure glaucoma, pigmentary glaucoma,
exfoliation glaucoma, developmental glaucoma, secondary glaucoma,
phacogenic glaucoma, glaucoma secondary to intraocular hemorrhage,
traumatic glaucoma, neovascular glaucoma, drug-induced glaucoma,
toxic glaucoma, and glaucoma associated with intraocular tumors,
retinal detachments, severe chemical burns of the eye, and iris
atrophy.
In other embodiments, the neurodegenerative disease, disorder, or
condition is or is associated with a disease, disorder, or
condition of the nervous system selected from the group consisting
of amyotrophic lateral sclerosis (ALS), trigeminal neuralgia,
glossopharyngeal neuralgia, Bell's Palsy, myasthenia gravis,
muscular dystrophy, progressive muscular atrophy, primary lateral
sclerosis (PLS), pseudobulbar palsy, progressive bulbar palsy,
spinal muscular atrophy, inherited muscular atrophy, invertebrate
disk syndromes, cervical spondylosis, plexus disorders, thoracic
outlet destruction syndromes, peripheral neuropathies, prophyria,
Alzheimer's disease, Huntington's disease, Parkinson's disease,
Parkinson's-plus diseases, multiple system atrophy, progressive
supranuclear palsy, corticobasal degeneration, dementia with Lewy
bodies, frontotemporal dementia, demyelinating diseases,
Guillain-Barre syndrome, multiple sclerosis, Charcot-Marie-Tooth
disease, prion diseases, Creutzfeldt-Jakob disease,
Gerstmann-Straussler-Scheinker syndrome (GSS), fatal familial
insomnia (FFI), bovine spongiform encephalopathy (BSE), Pick's
disease, epilepsy, AIDS demential complex, alcoholism, Alexander's
disease, Alper's disease, ataxia telangiectasia, Batten disease,
Canavan disease, Cockayne syndrome, diabetic neuropathy,
frontotemporal lobar degeneration, HIV-associated dementia,
Kennedy's disease, Krabbe's disease, neuroborreliosis,
Machado-Joseph disease (Spinocerebellar ataxia type 3), wet or dry
macular degeneration, Niemann Pick disease, Pelizaeus-Merzbacher
Disease, photoreceptor degenerative diseases, Refsum's disease,
Sandhoffs disease, Schilder's disease, subacute combined
degeneration of spinal cord secondary to pernicious anemia,
Spielmeyer-Vogt-Sjogren-Batten disease (also known as Batten
disease), spinocerebellar ataxia (multiple types with varying
characteristics), Steele-Richardson-Olszewski disease, and tabes
dorsalis.
In yet other embodiments, the neurodegenerative disease, disorder,
or condition comprises one or more conditions that are secondary to
a disease, disorder, condition, or therapy having a primary effect
outside of the nervous system selected from the group consisting
of: peripheral neuropathy or neuralgia caused by diabetes, cancer,
AIDS, hepatitis, kidney dysfunction, Colorado tick fever,
diphtheria, HIV infection, leprosy, Lyme disease, polyarteritis
nodosa, rheumatoid arthritis, sarcoidosis, Sjogren syndrome,
syphilis, systemic lupus erythematosus, and amyloidosis.
In other embodiments, the neurodegenerative disease, disorder, or
condition is associated with pain selected from the group
consisting of chronic pain, fibromyalgia, spinal pain, carpel
tunnel syndrome, pain from cancer, arthritis, sciatica, headaches,
pain from surgery, muscle spasms, back pain, visceral pain, pain
from injury, dental pain, neuralgia, such as neurogenic or
neuropathic pain, nerve inflammation or damage, shingles, herniated
disc, a torn ligament, and diabetes.
In further embodiments, the neurodegenerative disease, disorder, or
condition is associated with one or more injuries to the nervous
system. In particular embodiments, the one or more injuries to the
nervous system is related to nerve damage caused by exposure to one
or more agents selected from the group consisting of toxic
compounds, heavy metals, industrial solvents, drugs,
chemotherapeutic agents, dapsone, HIV medications, cholesterol
lowering drugs, heart or blood pressure medications, and
metronidazole.
In more particular embodiments, the one or more injuries to the
nervous system is related to nerve damage caused by one or more
conditions selected from the group consisting of burn, wound,
surgery, accidents, ischemia, prolonged exposure to cold
temperature, stroke, intracranial hemorrhage, and cerebral
hemorrhage.
In yet other embodiments, the neurodegenerative disease, disorder,
or condition comprises a psychiatric disorder. In particular
embodiments, the psychiatric disorder is selected from the group
consisting of schizophrenia, delusional disorder, schizoaffective
disorder, schizopheniform, shared psychotic disorder, psychosis,
paranoid personality disorder, schizoid personality disorder,
borderline personality disorder, anti-social personality disorder,
narcissistic personality disorder, obsessive-compulsive disorder,
delirium, dementia, mood disorders, bipolar disorder, depression,
stress disorder, panic disorder, agoraphobia, social phobia,
post-traumatic stress disorder, anxiety disorder, and impulse
control disorders.
In some embodiments, the method promotes or stimulates neurite
growth or regeneration from one or more neuronal cells.
In further embodiments, the method comprises treating one or more
neuronal cells in preparation for a nerve transplantation
procedure. In particular embodiments, the treating is before,
during, or after the transplantation procedure.
In other embodiments, the method treats or prevents a neuronal cell
loss in the subject. In yet other embodiments, the method prevents
neuronal cell death in the subject. In some embodiments, the method
prevents apoptosis of one or more neuronal axons in the
subject.
In some embodiments, an additional therapeutic agent is
administered to the subject. In particular embodiments, the
additional therapeutic agent is selected from the group consisting
of a beta-blocker, an alpha-agonist, a carbonic anhydrase
inhibitor, a prostaglandin or a prostaglandin analog, a miotic or a
cholinergic agent, an epinephrine compound, forskolin, and one or
more additional neuroprotective compounds.
In some embodiments, the compound against the identified target, or
pathway, or a pharmaceutically acceptable salt thereof, is
administered to the subject by a method selected from the group
consisting of oral, topical, parenteral, and systemic.
In other embodiments, the presently disclosed subject matter
provides a method for treating or preventing a neurodegenerative
disease, disorder, or condition in a subject in need thereof, the
method comprising administering to the subject a therapeutically
effective amount of a compound that modifies one of the described
molecular targets of associated signaling pathway, or a
pharmaceutically acceptable salt thereof, or a siRNA, shRNA, or
other gene expression or kinase or pathway modifying agent, thereby
treating or preventing the neurodegenerative disease, disorder, or
condition.
As used herein, a "neuron or portion thereof" can consist of or be
a portion of a neuron selected from the group consisting of a
cerebellar granule neuron, a dorsal root ganglion neuron, a
cortical neuron, a sympathetic neuron, and a hippocampal neuron.
More particularly, the term "neuron" as used herein denotes nervous
system cells that include a central cell body or soma, and two
types of extensions or projections: dendrites, by which, in
general, the majority of neuronal signals are conveyed to the cell
body; and axons, by which, in general, the majority of neuronal
signals are conveyed from the cell body to effector cells, such as
target neurons or muscle. Neurons can convey information from
tissues and organs into the central nervous system (afferent or
sensory neurons) and transmit signals from the central nervous
systems to effector cells (efferent or motor neurons). Other
neurons, designated interneurons, connect neurons within the
central nervous system (the brain and spinal column) Certain
specific examples of neuron types that may be subject to treatment
according to the presently disclosed subject matter include
cerebellar granule neurons, dorsal root ganglion neurons, and
cortical neurons. Further, as used herein, the term "neurite" means
a projection from the cell body of a neuron including, e.g., an
axon or a dendrite.
Without wishing to be bound to any one particular theory, it is
believed that the presently disclosed targets and pathways can
modulate: (i) the activity or expression of a target protein in the
neuron or portion thereof; (ii) a process in the neuron or portion
thereof; or (iii) a biological pathway associated with a
neurodegenerative disease, disorder, or condition. In particular
embodiments, the presently disclosed compounds inhibit one or more
protein kinases involved in a biological pathway associated with a
neurodegenerative disease, disorder, or condition. As used herein,
the term "inhibit" or "inhibits" means to decrease, suppress,
attenuate, diminish, arrest, or stabilize the development or
progression of a disease, disorder, or condition, or the activity
of a biological pathway, e.g., by at least 10%, 20%, 30%, 40%, 50%,
60%, 70%, 80%, 90%, 95%, 98%, 99%, or even 100% compared to an
untreated control subject, cell, or biological pathway. By the term
"decrease" is meant to inhibit, suppress, attenuate, diminish,
arrest, or stabilize a symptom of a neurodegenerative disease,
disorder, or condition. It will be appreciated that, although not
precluded, treating a disease, disorder or condition does not
require that the disease, disorder, condition or symptoms
associated therewith be completely eliminated.
In some embodiments, the neuron or portion thereof can be present
in a subject, such as a human subject. The subject can, for
example, have or be at risk of developing a disease, disorder, or
condition selected from the group consisting of (i) a disease,
disorder, or condition of the nervous system; (ii) a condition of
the nervous system that is secondary to a disease, disorder, or
condition, or a therapy having a primary effect outside of the
nervous system; (iii) an injury to the nervous system, such as, for
example, an injury caused by physical, mechanical, or chemical
trauma; (iv) pain; (v) ocular-related neurodegeneration; (vi)
memory loss; and (vii) a psychiatric disorder.
Accordingly, in some embodiments, a compound against one of the
identified targets, or pathways, can be used to treat or prevent a
neurodegenerative disease, disorder, or condition. As used herein,
the terms "treat," "treating," "treatment," and the like, are meant
to decrease, suppress, attenuate, diminish, arrest, the underlying
cause of a disease, disorder, or condition, or to stabilize the
development or progression of a disease, disorder, condition,
and/or symptoms associated therewith. The terms "treat,"
"treating," "treatment," and the like, as used herein can refer to
curative therapy, prophylactic therapy, and preventative therapy.
The treatment, administration, or therapy can be consecutive or
intermittent. Consecutive treatment, administration, or therapy
refers to treatment on at least a daily basis without interruption
in treatment by one or more days. Intermittent treatment or
administration, or treatment or administration in an intermittent
fashion, refers to treatment that is not consecutive, but rather
cyclic in nature. Treatment according to the presently disclosed
methods can result in complete relief or cure from a disease,
disorder, or condition, or partial amelioration of one or more
symptoms of the disease, disease, or condition, and can be
temporary or permanent. The term "treatment" also is intended to
encompass prophylaxis, therapy and cure.
As used herein, the terms "prevent," "preventing," "prevention,"
"prophylactic treatment" and the like refer to reducing the
probability of developing a disease, disorder, or condition in a
subject, who does not have, but is at risk of or susceptible to
developing a disease, disorder, or condition. Thus, in some
embodiments, an agent can be administered prophylactically to
prevent the onset of a disease, disorder, or condition, or to
prevent the recurrence of a disease, disorder, or condition.
By "agent" is meant a compound against one of the identified
targets, or pathways, or another agent, e.g., a peptide, nucleic
acid molecule, or other small molecule compound administered in
combination with a compound that modulates the expression or
activity of one of the identified targets or pathways. More
generally, the term "therapeutic agent" means a substance that has
the potential of affecting the function of an organism. Such an
agent may be, for example, a naturally occurring, semi-synthetic,
or synthetic agent. For example, the therapeutic agent may be a
drug that targets a specific function of an organism. A therapeutic
agent also may be a nutrient. A therapeutic agent may decrease,
suppress, attenuate, diminish, arrest, or stabilize the development
or progression of disease, disorder, or condition in a host
organism.
The term "administering" as used herein refers to contacting a
neuron or portion thereof with a compound against one of the
identified targets, or pathways. This term includes administration
of the presently disclosed compounds to a subject in which the
neuron or portion thereof is present, as well as introducing the
presently disclosed compounds into a medium in which a neuron or
portion thereof is cultured.
By "neurodegenerative disease, disorder, or condition" is meant a
disease, disorder, or condition (including a neuropathy) associated
with degeneration or dysfunction of neurons or other neural cells,
such as retinal ganglion cells. A neurodegenerative disease,
disorder, or condition can be any disease, disorder, or condition
in which decreased function or dysfunction of neurons, or loss or
neurons or other neural cells, can occur. Particular targets
associated with neurodegenerative diseases, disorders, or
conditions are disclosed in International PCT Patent Application
Publication No. WO2011/050192 to Lewcock et al., published Apr. 28,
2011, which is incorporated herein by reference in its
entirety.
As used herein, the term "disorder" in general refers to any
condition that would benefit from treatment with a compound against
one of the identified targets, or pathways, including any disease,
disorder, or condition that can be treated by an effective amount
of a compound against one of the identified targets, or pathways,
or a pharmaceutically acceptable salt thereof.
Such diseases, disorders, or conditions include, but are not
limited to, glaucoma, and neurodegenerative diseases, disorders, or
conditions of the nervous systems, such as or associated with
amyotrophic lateral sclerosis (ALS), trigeminal neuralgia,
glossopharyngeal neuralgia, Bell's Palsy, myasthenia gravis,
muscular dystrophy, progressive muscular atrophy, primary lateral
sclerosis (PLS), pseudobulbar palsy, progressive bulbar palsy,
spinal muscular atrophy, inherited muscular atrophy, invertebrate
disk syndromes, cervical spondylosis, plexus disorders, thoracic
outlet destruction syndromes, peripheral neuropathies, prophyria,
Alzheimer's disease, Huntington's disease, Parkinson's disease,
Parkinson's-plus diseases, multiple system atrophy, progressive
supranuclear palsy, corticobasal degeneration, dementia with Lewy
bodies, frontotemporal dementia, demyelinating diseases,
Guillain-Barre syndrome, multiple sclerosis, Charcot-Marie-Tooth
disease, prion diseases, Creutzfeldt-Jakob disease,
Gerstmann-Straussler-Scheinker syndrome (GSS), fatal familial
insomnia (FFI), bovine spongiform encephalopathy (BSE), Pick's
disease, epilepsy, and AIDS demential complex.
Other neurodegenerative diseases, disorders, or conditions of the
nervous systems, such as or associated with alcoholism, Alexander's
disease, Alper's disease, ataxia telangiectasia, Batten disease
(also known as Spielmeyer-Vogt-Sjogren-Batten disease), Canavan
disease, Cockayne syndrome, diabetic neuropathy, frontotemporal
lobar degeneration, HIV-associated dementia, Kennedy's disease,
Krabbe's disease, neuroborreliosis, Machado-Joseph disease
(Spinocerebellar ataxia type 3), wet or dry macular degeneration,
Niemann Pick disease, Pelizaeus-Merzbacher Disease, photoreceptor
degenerative diseases, such as retinitis pigmentosa and associated
diseases, Refsum's disease, Sandhoffs disease, Schilder's disease,
subacute combined degeneration of spinal cord secondary to
pernicious anemia, Spielmeyer-Vogt-Sjogren-Batten disease (also
known as Batten disease), spinocerebellar ataxia (multiple types
with varying characteristics), Steele-Richardson-Olszewski disease,
and tabes dorsalis.
Examples of ocular-related neurodegeneration include, but are not
limited to, glaucoma, lattice dystrophy, retinitis pigmentosa,
age-related macular degeneration (AMD), photoreceptor degeneration
associated with wet or dry AMD, other retinal degeneration, optic
nerve drusen, optic neuropathy, and optic neuritis, such as optic
neuritis resulting from multiple sclerosis.
Non-limiting examples of different types of glaucoma that can be
prevented or treated according to the presently disclosed subject
matter include primary glaucoma (also known as primary open-angle
glaucoma, chronic open-angle glaucoma, chronic simple glaucoma, and
glaucoma simplex), low-tension glaucoma, primary angle-closure
glaucoma (also known as primary closed-angle glaucoma, narrow-angle
glaucoma, pupil-block glaucoma, and acute congestive glaucoma),
acute angle-closure glaucoma, chronic angle-closure glaucoma,
intermittent angle-closure glaucoma, chronic open-angle closure
glaucoma, pigmentary glaucoma, exfoliation glaucoma (also known as
pseudoexfoliative glaucoma or glaucoma capsulare), developmental
glaucoma (e.g., primary congenital glaucoma and infantile
glaucoma), secondary glaucoma (e.g., inflammatory glaucoma (e.g.,
uveitis and Fuchs heterochromic iridocyclitis)), phacogenic
glaucoma (e.g., angle-closure glaucoma with mature cataract,
phacoanaphylactic glaucoma secondary to rupture of lens capsule,
phacolytic glaucoma due to phacotoxic meshwork blockage, and
subluxation of lens), glaucoma secondary to intraocular hemorrhage
(e.g., hyphema and hemolytic glaucoma, also known as erythroclastic
glaucoma), traumatic glaucoma (e.g., angle recession glaucoma,
traumatic recession on anterior chamber angle, postsurgical
glaucoma, aphakic pupillary block, and ciliary block glaucoma),
neovascular glaucoma, drug-induced glaucoma (e.g., corticosteroid
induced glaucoma and alpha-chymotrypsin glaucoma), toxic glaucoma,
and glaucoma associated with intraocular tumors, retinal
detachments, severe chemical burns of the eye, and iris atrophy. In
certain embodiments, the neurodegenerative disease, disorder, or
condition is a disease, disorder, or condition that is not
associated with excessive angiogenesis, for example, a glaucoma
that is not neovascular glaucoma.
Examples of conditions of the nervous system that are secondary to
a disease, disorder, condition, or therapy having a primary effect
outside of the nervous system include, but are not limited to,
peripheral neuropathy or neuralgia caused by diabetes, cancer,
AIDS, hepatitis, kidney dysfunction, Colorado tick fever,
diphtheria, HIV infection, leprosy, Lyme disease, polyarteritis
nodosa, rheumatoid arthritis, sarcoidosis, Sjogren syndrome,
syphilis, systemic lupus erythematosus, and amyloidosis.
Examples of pain include, but are not limited to, chronic pain,
fibromyalgia, spinal pain, carpel tunnel syndrome, pain from
cancer, arthritis, sciatica, headaches, pain from surgery, muscle
spasms, back pain, visceral pain, pain from injury, dental pain,
neuralgia, such as neurogenic or neuropathic pain, nerve
inflammation or damage, shingles, herniated disc, a torn ligament,
and diabetes.
Examples of injuries to the nervous system caused by physical,
mechanical, or chemical trauma include, but are not limited to,
nerve damage caused by exposure to toxic compounds, heavy metals
(e.g., lead, arsenic, and mercury), industrial solvents, drugs,
chemotherapeutic agents, dapsone, HIV medications (e.g.,
zidovudine, didanosine, stavudine, zalcitabine, ritonavir, and
amprenavir), cholesterol lowering drugs (e.g., lovastatin,
indapamide, and gemfibrozil), heart or blood pressure medications
(e.g., amiodarone, hydralazine, perhexyline), and
metronidazole.
Further examples also include burn, wound, surgery, accidents,
ischemia, prolonged exposure to cold temperature (e.g., frost
bite), stroke, intracranial hemorrhage, and cerebral hemorrhage.
More particularly, traumatic injury or other damage to neuronal
cells (e.g., trauma due to accident, blunt-force injury, gunshot
injury, spinal cord injury, ischemic conditions of the nervous
system such as stroke, cell damage due to aging or oxidative
stress, and the like) also is intended to be included within the
language "neurodegenerative disease, disorder, or condition." In
such embodiments, the presently disclosed methods can be used to
treat neuronal damage due to traumatic injury or stroke by
preventing death of damaged neuronal cells and/or by promoting or
stimulating neurite growth from damaged neuronal cells.
Examples of psychiatric disorders include, but are not limited to,
schizophrenia, delusional disorder, schizoaffective disorder,
schizopheniform, shared psychotic disorder, psychosis, paranoid
personality disorder, schizoid personality disorder, borderline
personality disorder, anti-social personality disorder,
narcissistic personality disorder, obsessive-compulsive disorder,
delirium, dementia, mood disorders, bipolar disorder, depression,
stress disorder, panic disorder, agoraphobia, social phobia,
post-traumatic stress disorder, anxiety disorder, and impulse
control disorders.
The subject treated by the presently disclosed methods in their
many embodiments is desirably a human subject, although it is to be
understood that the methods described herein are effective with
respect to all vertebrate species, which are intended to be
included in the term "subject." Accordingly, a "subject" can
include a human subject for medical purposes, such as for the
treatment of an existing disease, disorder, condition or the
prophylactic treatment for preventing the onset of a disease,
disorder, or condition or an animal subject for medical, veterinary
purposes, or developmental purposes. Suitable animal subjects
include mammals including, but not limited to, primates, e.g.,
humans, monkeys, apes, gibbons, chimpanzees, orangutans, macaques
and the like; bovines, e.g., cattle, oxen, and the like; ovines,
e.g., sheep and the like; caprines, e.g., goats and the like;
porcines, e.g., pigs, hogs, and the like; equines, e.g., horses,
donkeys, zebras, and the like; felines, including wild and domestic
cats; canines, including dogs; lagomorphs, including rabbits,
hares, and the like; and rodents, including mice, rats, guinea
pigs, and the like. An animal may be a transgenic animal. In some
embodiments, the subject is a human including, but not limited to,
fetal, neonatal, infant, juvenile, and adult subjects. Further, a
"subject" can include a patient afflicted with or suspected of
being afflicted with a disease, disorder, or condition. Thus, the
terms "subject" and "patient" are used interchangeably herein.
Subjects also include animal disease models (e.g., rats or mice
used in experiments, e.g., optic crush experiments, and the
like).
In particular embodiments, the subject is suffering from or
susceptible to a neurodegenerative disease, disorder, or condition,
such as glaucoma, e.g., a subject diagnosed as suffering from or
susceptible to a neurodegenerative disease, disorder, or condition.
In other embodiments, the subject has been identified (e.g.,
diagnosed) as suffering from or susceptible to a neurodegenerative
disease, disorder, or condition (including traumatic injury) in
which neuronal cell loss is implicated, or in which damage to
neurites is involved, and for which treatment or prophylaxis is
desired.
In certain embodiments, the subject is not suffering, or has not
been diagnosed as suffering, from cancer. In certain embodiments,
the subject is not suffering, or has not been diagnosed as
suffering, from a disorder related to excess angiogenesis. In
certain embodiments in which a cell is contacted with a compound
against one of the identified targets, or pathways, or a
pharmaceutically acceptable salt thereof, the cell is not a
neoplastic cell. In certain embodiments of the above aspects, the
cell is a mammalian cell, more preferably a human cell.
In some embodiments, the presently disclosed methods produce at
least about a 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,
60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or even 100% decrease in
cell loss or loss of function relative to cell survival or cell
function measured in absence of the tested compound, i.e., a
control sample or cell, in an assay. In other embodiments, the
compounds and amounts for use in the presently disclosed
therapeutic methods produce at least about 10% to 15% increase in
neuron count, neuron function, neurite count, neurite total length,
or neurite average length relative to absence of the tested
compound in an assay.
In any of the above-described methods, the administering of a
compound against one of the identified targets, or pathways, can
result in at least about a 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,
50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or even 100%
decrease in one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8. 9, or 10)
symptoms of a disease, disorder, or condition of the nervous
system; a condition of the nervous system that is secondary to a
disease, disorder, condition, or therapy having a primary effect
outside of the nervous system; injury to the nervous system caused
by physical, mechanical, or chemical trauma; pain; ocular-related
neurodegeneration; memory loss; or psychiatric disorder, compared
to a subject that is not administered the one or more of the agents
described herein.
Non-limiting examples of such symptoms include, but are not limited
to, tremors, slowness of movement, ataxia, loss of balance,
depression, decreased cognitive function, short-term memory loss,
long-term memory loss, confusion, changes in personality, language
difficulties, loss of sensory perception, sensitivity to touch,
numbness in extremities, muscle weakness, muscle paralysis, muscle
cramps, muscle spasms, significant changes in eating habits,
excessive fear or worry, insomnia, delusions, hallucinations,
fatigue, back pain, chest pain, digestive problems, headache, rapid
heart rate, dizziness, blurred vision, shadows or missing areas of
vision, metamorphopsia, impairment in color vision, decreased
recovery of visual function after exposure to bright light, and
loss in visual contrast sensitivity.
In any of the above-described methods, the administering of a
compound against one of the identified targets, or pathways,
results in at least about a 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,
50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or even 100%
decrease in the likelihood of developing a disease, disorder, or
condition of the nervous system; condition of the nervous system
that is secondary to a disease, disorder, condition, or therapy
having a primary effect outside of the nervous system; injury to
the nervous system caused by physical, mechanical, or chemical
trauma; pain; ocular-related neurodegeneration; memory loss; or
psychiatric disorder, compared to a control population of subjects
that are not administered a compound against one of the identified
targets, or pathways.
The administration of one or more agent as described herein may
result in at least about a 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,
50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or even 100%
decrease in the number of neurons (or neuron bodies, axons, or
dendrites thereof) that degenerate in a neuron population or in a
subject compared to the number of neurons (or neuron bodies, axons,
or dendrites thereof) that degenerate in neuron population or in a
subject that is not administered the one or more of the agents
described herein.
In some embodiments, the presently disclosed methods include
preventing or inhibiting neuron or axon degeneration. The phrases
"preventing axon degeneration," "preventing neuron degeneration,"
"inhibiting axon degeneration," or "inhibiting neuron degeneration"
as used herein include: (i) the ability to inhibit or prevent axon
or neuron degeneration in patients newly diagnosed as having a
neurodegenerative disease or at risk of developing a new
neurodegenerative disease; and (ii) the ability to inhibit or
prevent further axon or neuron degeneration in patients who are
already suffering from, or have symptoms of, a neurodegenerative
disease. Preventing axon or neuron degeneration includes decreasing
or inhibiting axon or neuron degeneration, which may be
characterized by complete or partial inhibition of neuron or axon
degeneration. Such prevention or inhibition can be assessed, for
example, by analysis of neurological function. Further, the phrases
"preventing neuron degeneration" and "inhibiting neuron
degeneration" include such inhibition with respect to the entire
neuron or a portion thereof, such as the neuron cell body, axons,
and dendrites.
The above-listed terms also include in vitro and ex vivo methods.
For example, in certain embodiments, the presently disclosed
methods are applicable to cell culture techniques wherein it is
desirable to prevent neuronal cell death or loss of neuronal
function. In vitro neuronal culture systems have proved to be
fundamental and indispensable tools for the study of neural
development, as well as the identification of neurotrophic factors,
such as nerve growth factor (NGF), ciliary trophic factors (CNTF),
and brain derived neurotrophic factor (BDNF). One use of certain
embodiments of the presently disclosed methods is in cultures of
neuronal cells, such as in the use of such cultures for the
generation of new neurons and glia. In such embodiments, the
cultured cells can be contacted with a compound against one of the
identified targets, or pathways, to prevent neuronal cell death or
loss of neuronal function. In an exemplary embodiment, the subject
method can be used to culture, for example, sensory neurons or,
alternatively, motoneurons. Such neuronal cultures can be used as
convenient assay systems, as well as sources of implantable cells
for therapeutic treatments.
In other examples, the neuron or portion thereof treated according
to the presently disclosed methods is ex vivo or in vitro.
Accordingly, the presently disclosed compounds can be useful as
components of culture media for use in culturing nerve cells in
vitro. More particularly, in certain embodiments, the presently
disclosed methods can be used to improve the survival or
integration of transplanted neuronal cells into a host subject
(e.g., through a nerve graft or nerve transplant). Thus, for
example, a subject receiving a transplant of neuronal cells can be
treated (before, during, or after the transplantation procedure)
with compounds according to the presently disclosed methods, to
prevent cell death of the transplanted cells (or host cells that
may be perturbed during the transplantation procedure), and/or to
promote the growth of neurites in the transplanted cells or the
host neuronal cells, and thereby promote integration of the
transplanted cells into the host nervous system.
The presently disclosed subject matter further provides methods of
modulating the growth, cell size, and/or proliferation of a neuron
(e.g., cerebellar granule neuron, a dorsal root ganglion neuron, a
cortical neuron, or a sympathetic neuron) by contacting a neuron
with a compound against one of the identified targets, or
pathways.
C. Combination Therapies
In certain embodiments, presently disclosed subject matter also
includes combination therapies. Depending on the particular
disease, disorder, or condition to be treated or prevented,
additional therapeutic agents, which are normally administered to
treat or prevent that condition, may be administered in combination
with the compounds of this disclosure. These additional agents may
be administered separately, as part of a multiple dosage regimen,
from the composition comprising a compound against one of the
identified targets, or pathways. Alternatively, these agents may be
part of a single dosage form, mixed together with the compound
against one of the identified targets, or pathways, in a single
composition.
By "in combination with" is meant the administration of a compound
against one of the identified targets, or pathways, with one or
more therapeutic agents either simultaneously, sequentially, or a
combination thereof. Therefore, a cell or a subject administered a
combination of a compound against one of the identified targets, or
pathways, can receive a compound against one of the identified
targets, or pathways, and one or more therapeutic agents at the
same time (i.e., simultaneously) or at different times (i.e.,
sequentially, in either order, on the same day or on different
days), so long as the effect of the combination of both agents is
achieved in the cell or the subject. When administered
sequentially, the agents can be administered within 1, 5, 10, 30,
60, 120, 180, 240 minutes or longer of one another. In other
embodiments, agents administered sequentially, can be administered
within 1, 5, 10, 15, 20 or more days of one another. Where the
compound against one of the identified targets, or pathways, and
one or more therapeutic agents are administered simultaneously,
they can be administered to the cell or administered to the subject
as separate pharmaceutical compositions, each comprising either a
compound against one of the identified targets, or pathways, or one
or more therapeutic agents, or they can contact the cell as a
single composition or be administered to a subject as a single
pharmaceutical composition comprising both agents. For example,
siRNA against MAP2K4 and MAP2K7 may be administered together. As
another example, small molecules that inhibit MAP3K12 and MAP3K13
may be administered together. Multiple therapeutic agents may be
administered together to modulate one target or multiple
targets.
When administered in combination, the effective concentration of
each of the agents to elicit a particular biological response may
be less than the effective concentration of each agent when
administered alone, thereby allowing a reduction in the dose of one
or more of the agents relative to the dose that would be needed if
the agent was administered as a single agent. The effects of
multiple agents may, but need not be, additive or synergistic. The
agents may be administered multiple times.
In such combination therapies, the therapeutic effect of the first
administered compound is not diminished by the sequential,
simultaneous or separate administration of the subsequent
compound(s).
A compound against one of the identified targets, or pathways, can
be used in therapy in combination with one or more other compounds
used to treat a neurodegenerative disease, disorder, or condition.
For example, a compound against one of the identified targets, or
pathways, can be co-administered in combination with one or more
other compounds, for example, at a ratio in the range of
1:1-1:5-5:1, 1:1-1:10-10:1, 1:1-1:25-25:1, 1:1-1:100-100:1,
1:1-1:1000-1000:1 or 1:1-1:10,000-10,000:1, and the like. For
example, in the treatment of glaucoma, other anti-glaucoma
medicaments can be used in combination with compounds against one
of the identified targets, or pathways, including, but not limited
to, beta-blockers, including levobunolol (BETAGAN), timolol
(BETIMOL, TIMOPTIC), betaxolol (BETOPTIC) and metipranolol
(OPTIPRANOLOL); alpha-agonists, such as apraclonidine (IOPIDINE)
and brimonidine (ALPHAGAN); carbonic anhydrase inhibitors, such as
acetazolamide, methazolamide, dorzolamide (TRUSOPT) and
brinzolamide (AZOPT); prostaglandins or prostaglandin analogs such
as latanoprost (XALATAN), bimatoprost (LUMIGAN) and travoprost
(TRAVATAN); miotic or cholinergic agents, such as pilocarpine
(ISOPTO CARPINE, PILOPINE) and carbachol (ISOPTO CARBACHOL);
epinephrine compounds, such as dipivefrin (PROPINE); forskolin; or
neuroprotective compounds, such as brimonidine and memantine. In
certain embodiments, the compound used in combination with a
compound against one of the identified targets, or pathways, is not
an anti-angiogenic agent, such as a steroid derivative, such as
2-methoxyestradiol or analogs or derivatives thereof. In other
embodiments, the additional therapeutic agent can be an
antibiotic.
The presently disclosed compounds against one of the identified
targets, or pathways, can be optionally combined with or
administered in concert with each other or other agents known to be
useful in the treatment of the relevant disease, disorder, or
condition. Thus, in the treatment of ALS, for example, the
presently disclosed compounds can be administered in combination
with Riluzole (RILUTEK), minocycline, insulin-like growth factor 1
(IGF-1), and/or methylcobalamin. In another example, in the
treatment of Parkinson's disease, the presently disclosed compounds
can be administered with L-dopa, dopamine agonists (e.g.,
bromocriptine, pergolide, pramipexole, ropinirole, cabergoline,
apomorphine, and lisuride), dopa decarboxylase inhibitors (e.g.,
levodopa, benserazide, and carbidopa), and/or MAO-B inhibitors
(e.g., selegiline and rasagiline). In a further example, in the
treatment of Alzheimer's disease, the presently disclosed compounds
can be administered with acetylcholinesterase inhibitors (e.g.,
donepezil, galantamine, and rivastigmine) and/or NMDA receptor
antagonists (e.g., memantine). The combination therapies can
involve concurrent or sequential administration, by the same or
different routes, as determined to be appropriate by those of skill
in the art. The presently disclosed subject matter also includes
pharmaceutical compositions and kits including combinations as
described herein.
In other embodiments, the presently disclosed subject matter
includes a combination therapy of administering a compound against
one or more of the identified targets, or pathways, in combination
with surgery, e.g., surgical relief of intraocular pressure, e.g.,
via trabeculectomy, laser trabeculoplasty, or drainage implants,
and the like.
In still other embodiments, the combination therapy may include
administering a compound against one or more of the identified
targets along with a transfection reagent, such as
Lipofectamine-2000 (L2K), a lipid mediated transfection
reagent.
D. Dosage and Mode of Administration
The presently disclosed pharmaceutical compositions can be
administered using a variety of methods known in the art depending
on the subject and the particular disease, disorder, or condition
being treated. The administering can be carried out by, for
example, intravenous infusion; injection by intravenous,
intraperitoneal, intracerebral, intramuscular, intraocular,
intraarterial or intralesional routes; or topical or ocular
application.
More particularly, as described herein, the presently disclosed
compounds can be administered to a subject for therapy by any
suitable route of administration, including orally, nasally,
transmucosally, ocularly, rectally, intravaginally, parenterally,
including intramuscular, subcutaneous, intramedullary injections,
as well as intrathecal, direct intraventricular, intravenous,
intra-articullar, intra-sternal, intra-synovial, intra-hepatic,
intralesional, intracranial, intraperitoneal, intranasal, or
intraocular injections, intracisternally, topically, as by powders,
ointments or drops (including eyedrops), including buccally and
sublingually, transdermally, through an inhalation spray, or other
modes of delivery known in the art. For example, for ocular
administration, an eyedrop formulation can include an effective
concentration of a compound against one of the identified targets,
or pathways, together with other components, such as buffers,
wetting agents and the like. Intravitreal injection also may be
employed to administer a presently disclosed compound to the
eye.
The phrases "systemic administration," "administered systemically,"
"peripheral administration" and "administered peripherally" as used
herein mean the administration of a compound, drug or other
material other than directly into the central nervous system, such
that it enters the patient's system and, thus, is subject to
metabolism and other like processes, for example, subcutaneous
administration.
The phrases "parenteral administration" and "administered
parenterally" as used herein mean modes of administration other
than enteral and topical administration, usually by injection, and
includes, without limitation, intravenous, intramuscular,
intarterial, intrathecal, intracapsular, intraorbital, intraocular,
intracardiac, intradermal, intraperitoneal, transtracheal,
subcutaneous, subcuticular, intraarticular, subcapsular,
subarachnoid, intraspinal and intrasternal injection and
infusion.
For intracerebral use, the compounds can be administered
continuously by infusion into the fluid reservoirs of the CNS,
although bolus injection may be acceptable. The presently disclosed
compounds can be administered into the ventricles of the brain or
otherwise introduced into the CNS or spinal fluid. Administration
can be performed by use of an indwelling catheter and a continuous
administration means such as a pump, or it can be administered by
implantation, e.g., intracerebral implantation of a
sustained-release vehicle. More specifically, the presently
disclosed compounds can be injected through chronically implanted
cannulas or chronically infused with the help of osmotic minipumps.
Subcutaneous pumps are available that deliver proteins through a
small tubing to the cerebral ventricles. Highly sophisticated pumps
can be refilled through the skin and their delivery rate can be set
without surgical intervention. Examples of suitable administration
protocols and delivery systems involving a subcutaneous pump device
or continuous intracerebroventricular infusion through a totally
implanted drug delivery system are those used for the
administration of dopamine, dopamine agonists, and cholinergic
agonists to Alzheimer's disease patients and animal models for
Parkinson's disease, as described by Harbaugh, J. Neural Transm.
Suppl. 24:271, 1987; and DeYebenes et al., Mov. Disord. 2: 143,
1987.
The presently disclosed pharmaceutical compositions can be
manufactured in a manner known in the art, e.g. by means of
conventional mixing, dissolving, granulating, dragee-making,
levitating, emulsifying, encapsulating, entrapping or lyophilizing
processes.
More particularly, pharmaceutical compositions for oral use can be
obtained through combination of active compounds with a solid
excipient, optionally grinding a resulting mixture, and processing
the mixture of granules, after adding suitable auxiliaries, if
desired, to obtain tablets or dragee cores. Suitable excipients
include, but are not limited to, carbohydrate or protein fillers,
such as sugars, including lactose, sucrose, mannitol, or sorbitol;
starch from corn, wheat, rice, potato, or other plants; cellulose,
such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium
carboxymethyl cellulose; and gums including arabic and tragacanth;
and proteins, such as gelatin and collagen; and
polyvinylpyrrolidone (PVP:povidone). If desired, disintegrating or
solubilizing agents, such as cross-linked polyvinyl pyrrolidone,
agar, alginic acid, or a salt thereof, such as sodium alginate,
also can be added to the compositions.
Dragee cores are provided with suitable coatings, such as
concentrated sugar solutions, which also can contain gum arabic,
talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol
(PEG), and/or titanium dioxide, lacquer solutions, and suitable
organic solvents or solvent mixtures. Dyestuffs or pigments can be
added to the tablets or dragee coatings for product identification
or to characterize the quantity of active compound, e.g., dosage,
or different combinations of active compound doses.
Pharmaceutical compositions suitable for oral administration
include push-fit capsule s made of gelatin, as well as soft, sealed
capsules made of gelatin and a coating, e.g., a plasticizer, such
as glycerol or sorbitol. The push-fit capsules can contain active
ingredients admixed with a filler or binder, such as lactose or
starches, lubricants, such as talc or magnesium stearate, and,
optionally, stabilizers. In soft capsules, the active compounds can
be dissolved or suspended in suitable liquids, such as fatty oils,
liquid paraffin, or liquid polyethylene glycols (PEGs), with or
without stabilizers. Stabilizers can be added as warranted.
In some embodiments, the presently disclosed pharmaceutical
compositions can be administered by rechargeable or biodegradable
devices. For example, a variety of slow-release polymeric devices
have been developed and tested in vivo for the controlled delivery
of drugs, including proteinacious biopharmaceuticals. Suitable
examples of sustained release preparations include semipermeable
polymer matrices in the form of shaped articles, e.g., films or
microcapsules. Sustained release matrices include polyesters,
hydrogels, polylactides (U.S. Pat. No. 3,773,919; EP 58,481),
copolymers of L-glutamic acid and gamma ethyl-L-glutamate (Sidman
et al., Biopolymers 22:547, 1983), poly
(2-hydroxyethyl-methacrylate) (Langer et al., J. Biomed. Mater.
Res. 15:167, 1981; Langer, Chem. Tech. 12:98, 1982), ethylene vinyl
acetate (Langer et al., Id), or poly-D-(-)-3-hydroxybutyric acid
(EP 133,988A).
Sustained release compositions also include liposomally entrapped
compounds, which can be prepared by methods known per se (Epstein
et al., Proc. Natl. Acad. Sci. U.S.A. 82:3688, 1985; Hwang et al.,
Proc. Natl. Acad. Sci. U.S.A. 77:4030, 1980; U.S. Pat. Nos.
4,485,045 and 4,544,545; and EP 102,324A). Ordinarily, the
liposomes are of the small (about 200-800 Angstroms) unilamelar
type in which the lipid content is greater than about 30 mol %
cholesterol, the selected proportion being adjusted for the optimal
therapy. Such materials can comprise an implant, for example, for
sustained release of the presently disclosed compounds, which, in
some embodiments, can be implanted at a particular, pre-determined
target site.
Pharmaceutical compositions for parenteral administration include
aqueous solutions of active compounds. For injection, the presently
disclosed pharmaceutical compositions can be formulated in aqueous
solutions, for example, in some embodiments, in physiologically
compatible buffers, such as Hank's solution, Ringer' solution, or
physiologically buffered saline. Aqueous injection suspensions can
contain substances that increase the viscosity of the suspension,
such as sodium carboxymethyl cellulose, sorbitol, or dextran.
Additionally, suspensions of the active compounds or vehicles
include fatty oils, such as sesame oil, or synthetic fatty acid
esters, such as ethyl oleate or triglycerides, or liposomes.
Optionally, the suspension also can contain suitable stabilizers or
agents that increase the solubility of the compounds to allow for
the preparation of highly concentrated solutions.
For nasal or transmucosal administration generally, penetrants
appropriate to the particular barrier to be permeated are used in
the formulation. Such penetrants are generally known in the
art.
For inhalation delivery, the agents of the disclosure also can be
formulated by methods known to those of skill in the art, and may
include, for example, but not limited to, examples of solubilizing,
diluting, or dispersing substances such as, saline, preservatives,
such as benzyl alcohol, absorption promoters, and
fluorocarbons.
Additional ingredients can be added to compositions for topical
administration, as long as such ingredients are pharmaceutically
acceptable and not deleterious to the epithelial cells or their
function. Further, such additional ingredients should not adversely
affect the epithelial penetration efficiency of the composition,
and should not cause deterioration in the stability of the
composition. For example, fragrances, opacifiers, antioxidants,
gelling agents, stabilizers, surfactants, emollients, coloring
agents, preservatives, buffering agents, and the like can be
present. The pH of the presently disclosed topical composition can
be adjusted to a physiologically acceptable range of from about 6.0
to about 9.0 by adding buffering agents thereto such that the
composition is physiologically compatible with a subject's
skin.
In other embodiments, the pharmaceutical composition can be a
lyophilized powder, optionally including additives, such as 1 mM-50
mM histidine, 0.1%-2% sucrose, 2%-7% mannitol at a pH range of 4.5
to 5.5 that is combined with buffer prior to use.
The presently disclosed subject matter also includes the use of a
compound against one of the identified targets, or pathways, in the
manufacture of a medicament for neuroprotection.
Regardless of the route of administration selected, the presently
disclosed compounds, which may be used in a suitable hydrated form,
and/or the pharmaceutical compositions are formulated into
pharmaceutically acceptable dosage forms such as described below or
by other conventional methods known to those of skill in the
art.
The term "effective amount," as in "a therapeutically effective
amount," of a therapeutic agent refers to the amount of the agent
necessary to elicit the desired biological response. As will be
appreciated by those of ordinary skill in this art, the effective
amount of an agent may vary depending on such factors as the
desired biological endpoint, the agent to be delivered, the
composition of the pharmaceutical composition, the target tissue or
cell, and the like. More particularly, the term "effective amount"
refers to an amount sufficient to produce the desired effect, e.g.,
to reduce or ameliorate the severity, duration, progression, or
onset of a disease, disorder, or condition (e.g., a disease,
condition, or disorder related to loss of neuronal cells or cell
function), or one or more symptoms thereof; prevent the advancement
of a disease, disorder, or condition, cause the regression of a
disease, disorder, or condition; prevent the recurrence,
development, onset or progression of a symptom associated with a
disease, disorder, or condition, or enhance or improve the
prophylactic or therapeutic effect(s) of another therapy.
Actual dosage levels of the active ingredients in the presently
disclosed pharmaceutical compositions can be varied so as to obtain
an amount of the active ingredient that is effective to achieve the
desired therapeutic response for a particular subject, composition,
route of administration, and disease, disorder, or condition
without being toxic to the subject. The selected dosage level will
depend on a variety of factors including the activity of the
particular compound employed, or salt thereof, the route of
administration, the time of administration, the rate of excretion
of the particular compound being employed, the duration of the
treatment, other drugs, compounds and/or materials used in
combination with the particular compound employed, the age, sex,
weight, condition, general health and prior medical history of the
patient being treated, and like factors well known in the medical
arts.
A physician or veterinarian having ordinary skill in the art can
readily determine and prescribe the effective amount of the
pharmaceutical composition required. For example, the physician or
veterinarian could start doses of the compounds against one of the
identified targets, or pathways, employed in the pharmaceutical
composition at levels lower than that required to achieve the
desired therapeutic effect and gradually increase the dosage until
the desired effect is achieved. Accordingly, the dosage range for
administration will be adjusted by the physician as necessary. It
will be appreciated that an amount of a compound required for
achieving the desired biological response, e.g., neuroprotective
activity, may be different from the amount of compound effective
for another purpose.
In general, a suitable daily dose of a compound against one of the
identified targets, or pathways, will be that amount of the
compound that is the lowest dose effective to produce a therapeutic
effect. Such an effective dose will generally depend upon the
factors described above. Generally, doses of the compounds against
one of the identified targets, or pathways, will range from about
0.0001 to about 1000 mg per kilogram of body weight of the subject
per day. In certain embodiments, the dosage is between about 1
.mu.g/kg and about 500 mg/kg, more preferably between about 0.015
mg/kg and about 50 mg/kg. For example, in certain embodiments, a
dose can be about 1, 5, 10, 15, 20, or 40 mg/kg/day.
If desired, the effective daily dose of the active compound can be
administered as two, three, four, five, six or more sub-doses
administered separately at appropriate intervals throughout the
day, optionally, in unit dosage forms.
E. Methods for Identifying Neuronal Injury and Associated Genes
The presently disclosed subject matter also provides methods for
identifying injury of a neuron, such as a retinal ganglion cell.
Injury can be identified by measuring the amount of the target
gene, protein, or pathway. For example, if MAP3K12 is the target,
the amount of MAP3K12 expression or activity in a cell can be
measured and compared to the MAP3K12 expression or activity in an
uninjured retinal ganglion cell. Other ways of detecting the amount
of MAP3K12 also fall within the scope of the presently disclosed
subject matter, such as detection of MAP3K12 outside of the cell
(for e.g., by immunological techniques), detection of
autoantibodies against MAP3K12, detection of a modified version of
the MAP3K12 protein that may be more specific to an injured cell
(such as a posttranslational modification), and the like.
Accordingly, in one embodiment the presently disclosed subject
matter provides a method for identifying injury of a neuron or
retinal pigment epithelial (RPE) cell, the method comprising:
measuring the amount of expression or activity of a target protein
kinase in the neuron or RPE cell; and determining if the amount of
protein kinase expression or activity in the neuron or RPE cell is
greater than the amount of protein kinase expression or activity in
a control neuron or RPE cell; wherein a determination that the
amount of protein kinase expression or activity is greater in the
neuron or RPE cell compared to the control neuron or RPE cell is
indicative of injury in the neuron. In a particular embodiment, the
target protein kinase is selected from the group consisting of
MAP3K12, MAP3K13, MAP2K4, and MAP2K7. In another particular
embodiment, the neuron and control neuron are retinal ganglion
cells or photoreceptor cells.
In another embodiment, a method is provided for identifying a gene
encoding a protein kinase associated with neuronal or retinal
pigment epithelial (RPE) cell injury or death. In one embodiment,
the method comprises: providing a neuron or RPE cell; contacting
the neuron or RPE cell with at least one silencing RNA (siRNA)
targeting a protein kinase in an amount sufficient to inhibit or
increase kinase activity of the protein kinase; and determining
whether the neuron or RPE cell survives; wherein a determination
that the neuron or RPE cell survives is an indication that the gene
encoding the protein kinase is associated with neuronal or RPE cell
injury or death. In a particular embodiment, the target protein
kinase is selected from the group consisting of MAP3K12, MAP3K13,
MAP2K4, and MAP2K7. In another particular embodiment, the neuron is
a retinal ganglion cell or a photoreceptor cell.
F. Kits or Pharmaceutical Systems
The presently disclosed compounds and compositions can be assembled
into kits or pharmaceutical systems for use in treating or
preventing neurodegenerative diseases, disorders, or conditions. In
some embodiments, the presently disclosed kits or pharmaceutical
systems include a compound against one of the identified targets,
or pathways, or pharmaceutically acceptable salts thereof. In
particular embodiments, the compounds against one of the identified
targets, or pathways, or a pharmaceutically acceptable salt
thereof, are in unit dosage form. In further embodiments, the
compound against one of the identified targets, or pathways, or a
pharmaceutically acceptable salt, can be present together with a
pharmaceutically acceptable solvent, carrier, excipient, or the
like, as described herein.
In some embodiments, the presently disclosed kits comprise one or
more containers, including, but not limited to a vial, tube,
ampule, bottle and the like, for containing the compound. The one
or more containers also can be carried within a suitable carrier,
such as a box, carton, tube or the like. Such containers can be
made of plastic, glass, laminated paper, metal foil, or other
materials suitable for holding medicaments.
In some embodiments, the container can hold a composition that is
by itself or when combined with another composition effective for
treating or preventing the condition and may have a sterile access
port (for example the container may be an intravenous solution bag
or a vial having a stopper pierceable by a hypodermic injection
needle). Alternatively, or additionally, the article of manufacture
may further include a second (or third) container including a
pharmaceutically-acceptable buffer, such as bacteriostatic water
for injection (BWFI), phosphate-buffered saline, Ringer's solution
and dextrose solution. It may further include other materials
desirable from a commercial and user standpoint, including other
buffers, diluents, filters, needles, and syringes.
The presently disclosed kits or pharmaceutical systems also can
include associated instructions for using the compounds for
treating or preventing a neurodegenerative disease, disorder, or
condition. In some embodiments, the instructions include one or
more of the following: a description of the active compound; a
dosage schedule and administration for treating or preventing a
neurodegenerative disease, disorder, or condition; precautions;
warnings; indications; counter-indications; overdosage information;
adverse reactions; animal pharmacology; clinical studies; and
references. The instructions can be printed directly on a container
(when present), as a label applied to the container, as a separate
sheet, pamphlet, card, or folder supplied in or with the
container.
EXAMPLES
The following Examples have been included to provide guidance to
one of ordinary skill in the art for practicing representative
embodiments of the presently disclosed subject matter. In light of
the present disclosure and the general level of skill in the art,
those of skill can appreciate that the following Examples are
intended to be exemplary only and that numerous changes,
modifications, and alterations can be employed without departing
from the scope of the presently disclosed subject matter. The
following Examples are offered by way of illustration and not by
way of limitation.
Example 1
Broad-Spectrum Kinase Inhibitors, Sunitinib and Vx680, are
Neuroprotective to Primary RGCS In Vitro and In Vivo
To determine the neuroprotective effects of the broad-spectrum
kinase inhibitors, sunitinib and VX680, primary RGCs were plated in
neurotrophin-depleted media and stained with calcein AM at 72 hours
in the absence (vehicle) or presence of 1 .mu.M sunitinib (FIG. 1,
Panel A). As can be seen from the higher number of stained cells in
the sunitinib sample, the survival of the RGCs was greatly
increased when sunitinib was added to the culture.
The survival of primary RGCs in response to increasing doses of
sunitinib and VX680 is shown in FIG. 1B. Both sunitinib and VX680
were neuroprotective to primary RGSs. However, at higher doses,
VX680 resulted in a greater number of viable cells whereas higher
doses of sunitinib resulted in more cell death.
To test the kinase inhibitors in vivo, C57Bl/6 mice were injected
intravitreally with 1 .mu.L of drug-eluting microspheres containing
0.125 mg/mL, 0.25 mg/mL, or 0.5 mg/mL of sunitinib, followed by
optic nerve crush one week later. RGC survival two weeks after
crush was assessed by Nissl staining and counting the nuclei in the
ganglion cell layer (GCL). Displaced amacrine cells, whose survival
is not affected by optic nerve crush, were assumed to represent 50%
of the GCL and thus a 20% reduction in nuclei was interpreted to be
a 40% reduction in RGCs. FIG. 1C shows that the addition of
sunitinib protected the ganglion cells in the mice as compared to
the control (vehicle).
Representative Nissl-stained sections of the GCL show the survival
benefit conferred by 0.5 mg/mL sustained-release sunitinib (Sutent)
(FIG. 1D).
Example 2
Highly Efficient Knockdown in Primary RGCS Using
Magnetofection-Based siRNA Delivery
FIG. 2A shows RGCs that were transfected at the time of isolation
with 20 nM siGLO-red with (bottom) or without (top) the
transfection reagent, NeuroMag and stained for viability at 24
hours with calcein AM. The magnetofection-based siRNA delivery
resulted in highly efficient knockdown in primary RGCs as seen by
the number of stained cells.
FIG. 2B shows RGCs that were transfected with increasing amounts of
siRNA against GAPDH (G) or a nontargeting control (C) and
immunoblotted for GAPDH or .beta.-tubulin (control) expression at
48 hours. Transfection with increasing amounts of siRNA resulted in
a decreasing amount of GAPDH protein, showing that the siRNA
transfection was effective.
Example 3
Screening a Kinase-Enriched siRNA Library in Primary RGCS
Identifies a Validated Neuroprotective Target
RGCs were plated in a 96-well format and transfected with 2112
siRNAs (20 nM) targeting 704 genes. In addition, each 96-well plate
had 6 negative control wells (nontargeting siRNA) and 2 positive
control wells (toxic All-Stars #3 siRNA pool; Qiagen). Survival at
72 hours for any given well was normalized to the mean of the 6
control wells (defined as 1.0) (FIG. 3A).
FIG. 3B shows the normalized survival of the top three candidates,
defined as genes with 2/3 or 3/3 siRNAs with greater than 1.30
normalized survival (3 SD from mean). These candidates were
MAP3K12, MAP2K4, and MAP2K7.
A confirmation screen was performed using an independent set of
siGenome or On-Target plus siRNAs (Dharmacon). MAP3K13 was not
included in the initial screen but was evaluated here given its
high degree of homology to MAP3K12. This screen showed that
knock-down of MAP3K12, MAP3K13, MAP2K4, and MAP2K7 promoted RGC
survival (FIG. 3C).
To further characterize these candidates, primary RGCs were
transfected with siRNAs against the various MAP kinases alone or in
combination (10 nM each) and survival was measured at 72 hours with
CellTiter-Glo. VX680 (1000 nM) is shown as a reference. A
photomicrograph of calcein AM-stained cells at 72 hours transfected
with control siRNA or siRNAs targeting MAP3K12 and MAP3K13 is also
shown (inset). These results showed that knock-down of MAP3K12,
MAP3K13, MAP2K4, and MAP2K7 promoted RGC survival (FIG. 3D).
RGCs transfected with siRNAs against both MAP3K12 and MAP3K13 were
followed over time with calcein AM staining. These results showed
that siRNA transfection against either MAP3K12, MAP3K13, or a
combination of both, resulted in higher survival as compared to the
control (FIG. 3E).
RGCs were transfected with 10 nM siRNA against MAP3K12 and MAP3K13
alone or in combination and then plated in 0, 12, 37, 111, 333, and
1000 nM sunitinib (FIG. 3F on left) or VX680 (FIG. 3F on right).
Survival at 72 hours is shown. These results showed that addition
of sunitinib or VX680 resulted in higher cell viability.
Example 4
MAP3K12/DLK is Upregulated in Response to Retinal Ganglion Cell
Injury
RGCs were immunopanned and then plated in 4-well format (250,000
cells per well). At the indicated times, cells were harvested and
MAP3K12 protein (FIG. 4A at top) and mRNA (FIG. 4A at bottom) were
measured by immunoblotting and quantitative RT-PCR, respectively.
GAPDH was used as loading control for both. These results showed
that MAP3K12 protein expression was stimulated by optic nerve
injury, with only a minimal change in RNA levels, indicating that
regulation was post-transcriptional.
C57Bl/6 mice were subjected to unilateral optic nerve crush (3
sec). Five days later, the mice were sacrificed and the retinas
were cryosectioned and stained for DAPI as a control (FIG. 4B at
top) or MAP3K12/DLK (FIG. 4B at bottom). Shown is a representative
section. Addition of MAP3K12/DLK siRNA resulted in increased
retinal ganglion cell viability.
Example 5
Knock-Down of MAP3K12/DLK Promotes RGC Survival and Neurite
Outgrowth
RGCs were transfected with MAP3K12/DLK siRNA and cultured for 14
days. The cells were then stained with calcein AM. Results
demonstrate that knock-down of MAP3K12/DLK promotes RGC survival
and neurite outgrowth. Cells without knock-down of MAP3K12/DLK
would essentially be dead at this time point.
Example 6
Knock-Down of Other Protein Kinases Promotes RGC Survival and
Neurite Outgrowth
Other protein kinases were also tested in this screen. RGCs were
transfected with each siRNA and cultured for 14 days. The cells
were then stained with calcein AM. Results demonstrated that
knock-down of a number of protein kinases promotes RGC survival and
neurite outgrowth. The protein kinases tested and found to promote
RGC survival were MAP3K12, MAP3K13, MAP3K14, MAP2K7, MAP2K4, LYN,
PLK3, PFKP, MARK3, MARK2, TAOK1, IKBKB, BRSK2, PBK, PRKCH, TESK1,
Csnk1e, Oxsr1, Tgfbr2, Mapk10, PFTK1, ERN2, AK2, HSPB8, FRAP1,
DGUOK, ERN1, STK32B, PIK3C2G, BCR, DYRK1A, DYRK1B, PNCK, EIF2AK1,
PKD2L1, NRK, Endothelin Receptor Type B, and TLK2.
The kinases listed, when inhibited by siRNA treatment,
substantially altered RGC neuron survival, indicating that they and
the pathways they regulate are targets for therapeutic
intervention. This may be by activating or inhibiting the target
gene by administration of siRNA or other gene expression or
activity modifying agents.
Example 7
Functional Genomic Screening Identifies MAP3K12/DLK as a Mediator
of Retinal Ganglion Cell Death
To identify kinases that could serve as novel targets for
neuroprotective glaucoma therapy, the entire mouse kinome was
screened for kinases whose inhibition promotes RGC survival. For
this screen, a high-throughout method was developed for
transfecting primary RGCs with small interfering RNA
oligonucleotides (siRNAs) and coupled with a quantitative assay of
RGC survival. Results of the screen are presented, which identified
the dual leucine zipper kinase (DLK; MAP3K12) as being important in
INK activation and RGC cell death following injury. Moreover it is
demonstrated, using a conditional knockout approach, that
inhibition of MAP3K12/DLK signaling can promote RGC survival in
vivo, and also identify a small molecule kinase inhibitor that
protects RGC somata and axons in a rodent model of glaucoma.
Materials and Methods
Statistical Analysis. All statistical analyses were performed with
the unpaired Mann-Whitney-Wilcoxon test.
Rat optic nerve transection. The optic nerve was exposed by a
partial peritomy and intraorbital dissection of the extraocular
muscles, and then transected with a 25-gauge needle. 4-Di-10-ASP
was then applied to the proximal nerve stump. Care was taken to
avoid vascular injury during the transection, and retinal perfusion
was examined after nerve transection. Two weeks after transection,
rats were sacrificed and enucleated. Retinas were flatmounted,
imaged with Zeiss LSM 510 META confocal microscope with a Zeiss
Plan-Apochromat 20.times./0.75 NA objective. Images were taken from
four fields of 230 mm.times.230 mm squares located 2 mm superior,
inferior, temporal, and nasal to the optic disc. The number of
4-Di-10-ASP-labeled cells with RGC morphology was quantified.
Imaging and quantification of RGC survival was performed in a
masked fashion.
Rat laser-induced ocular hypertension. Intraocular pressure (TOP)
was unilaterally elevated by laser treatment of the trabecular
meshwork as previously described (Levkovitch-Verbin et al.
Investigative Ophthalmology & Visual Science 43:402-410, 2002).
Briefly, 6-week old Wistar male rats were anesthetized with
ketamine/xylazine. On two consecutive weeks, 40-50 532 nm diode
laser spots were applied to the prelimbal region (50 .mu.m
diameter, 600 mW power and 0.6 seconds duration). Under anesthesia,
the IOP of laser treated and fellow eyes was measured with TonoLab
one and three days after laser treatment. Four weeks following
laser treatment, toluidine blue-stained optic nerves were imaged
and the number of axons counted. The laser treatment and
acquisition of optic nerve images were performed in a masked
fashion. RGC somata were measured by Brn3 staining of retina
sections. The number of Brn3-positive cells was normalized by the
number of DAPI-stained cells in GCL on the same sections.
Mouse intravitreal injection and optic nerve crush. 3-month old
male C57BL/6 and Dlk floxed mice (BL/6 background) were
anesthetized with ketamine/xylazine and intravitreally injected
with 1010 DNA-containing particles of capsid-mutant (Y444, 500,
730F) AAV2 expressing Cre recombinase from the chicken .beta.-actin
promoter. 7 days later, optic nerve was surgically exposed and
crushed with Dumont N7 self-closing forceps 1 mm behind the globe
for 3 seconds. 10 days following nerve crush, eyes were enucleated,
fixed and surviving RGC was immunostained for .beta.III-tubulin and
Brn3. The retinas were then imaged with a Nikon Eclipse TE2000-5
fluorescence microscope and Plan-fluor 40.times./0.6 objective.
Images were acquired from the four fields in the superior,
inferior, temporal and nasal quadrants 1 mm from the optic disc.
RGCs were counted manually from each image.
In a separate cohort of animals, optic nerves and retinas were
sectioned and stained for phospho-JNK and c-Jun, respectively, 24
hours after optic nerve crush. Intravitreal injection, optic nerve
crush, immunofluorescence and RGC counting were performed in a
masked fashion.
Reagents. Antibodies: Phospho-JNK, Thr183/Tyr185 (4671); JNK
(9258); c-Jun (9165); Phospho-MKK7 (4171) and MKK7 (4172) (Cell
Signaling Technology); monoclonal anti-alpha-tubulin antibody
(T6074) (Sigma); mouse neuronal class .beta.III tubulin (TUJ1)
(Covance); goat polyclonal Brn3 (C-13); and rabbit anti-Cre
(Novus).
Retinal Ganglion Cell Purification, Culture, Screening, and
Imaging. All animal use was in accordance with the Association for
Research in Vision and Opthalmology (ARVO) Statement for the Use of
Animals, following animal protocols approved by the Institutional
Animal Care and Use Committee at Johns Hopkins University. Retinas
were isolated from postnatal 0-5-d mice and dissociated with
papain. Microglia were immunodepleted with anti-CD11b conjugated
Dynabeads (Life Technologies). The suspension of retinal cells was
immunopanned on plates preconjugated with anti-Thy1.2 antibody
(Serotec, MCA028) and goat antimouse IgM (Jackson Immunoresearch)
at room temperature (RT). After washing, retinal ganglion cellS
(RGCs) were released from the plate by a cell lifter, counted, and
seeded at a density of 10,000 per well in 96-well plates in the
media composed of Neurobasal (Life Technologies), B27, N2
supplement, Lglutamine, and penicillin/streptomycin. After a 72-h
culture at 37.degree. C., RGCs were stained with calceinAM
(acetomethoxy derivate of calcein), ethidium homodimer, and Hoechst
33342. Images were taken from portions of each well with a
Cellomics ArrayScan VTI HCS Reader (Thermo Fisher), and cell
survival was quantified using the Cellomics Neuronal Profiling
bioapplication. As indicated, RGC viability was alternatively
measured by Cell-Titer-Glo (Promega) luminescence.
For siRNA-based screening, the siRNAs from the Sigma Mission Mouse
Kinome library were complexed with NeuroMag (Oz Biosciences) at a
final concentration of 20 nM. RGCs were then reverse transfected on
a stationary magnet and assayed for survival 72 h later using
CellTiter-Glo (Promega). Confirmatory siRNAs were obtained from
both Dharmacon and Ambion. Adenoviruses expressing wild-type or
kinase-dead dual leucine zipper kinase, GFP, or Cre were added to
RGCs at a multiplicity of infection of 100-1000.
Rat Intravitreal Injections. Six-week old male Wistar rats were
anesthetized with ketamine/xylazine. A partial peritomy was made to
expose the sclera. The injection site was approximately at the ora
serrata, and the injection glass pipet was angled toward the optic
disk to avoid lens injury. Five microliters (10 ng) of PLGA
microspheres were injected with a glass pipet and Hamilton
syringe.
Electrophysiology. Recordings were made by using the whole-cell
patch-clamp technique in both current- and voltage-clamp modes with
an Axopatch 200B (Molecular Devices). Data were low-pass filtered
at 1 kHz (Bessel) and sampled at 10 kHz. A liquid junction
potential of -2 mV has been corrected, and the resting potential
was estimated to be -62.+-.2.2 mV (mean.+-.SEM; n=13). The
recording pipette was filled with the following intracellular
solution (in mM): 100 K-gluconate, 50 KCl, 20 Hepes, 10 EGTA, 5
MgCl.sub.2, 2 ATP, 0.1 GTP, pH adjusted to 7.33 with KOH. The cells
were continuously perfused with (in mM) 140 NaCl, 5 KCl, 1
MgCl.sub.2, 2.5 CaCl.sub.2, 10 glucose, 10 Hepes, pH 7.4, with
NaOH.
Production of Adeno-Associated Virus Vectors. Adeno-associated
virus vector was produced by the 2-plasmid, cotransfection method
with modifications (Zolotukhin et al., Methods 28(2):158-167,
2002).
RNAi-Based Screen Identifies MAP3K12/DLK
In order to develop a biologically relevant RGC survival assay,
primary RGCs were immunopanned from perinatal mice (Barres et al.
Neuron 1:791-803, 1988). Despite the inherent challenges in the use
of primary neurons, they are more likely than established cell
lines to be predictive of in vivo efficacy (Sharma et al. Meth
Enzymol 506:331-360, 2012). Individually inhibiting the function of
each kinase in the genome required an efficient method for siRNA
delivery to the primary RGCs. Since traditional transfection
procedures were either toxic or minimally effective with RGCs, a
magnetic nanoparticle-based reagent (NeuroMag) was adapted for
high-efficiency, high-throughput siRNA delivery (FIG. 6A).
NeuroMag-based transfection resulted in consistent and efficient
suppression of target gene expression in unselected RGC populations
(FIG. 7).
Using this approach, an arrayed library of 1869 siRNAs was screened
against 623 kinases, providing three-fold coverage of the mouse
kinome, for the ability to promote the survival of RGCs grown in
neurotrophin-deficient media (FIG. 6B). To minimize the number of
false-positive leads resulting from off target silencing, a
conservative approach was taken to focus only on kinases for which
all three siRNAs were protective. Indeed, only two kinases met this
criterion, MAP3K12/DLK and its only known substrate,
mitogen-activated protein kinase kinase 7 (MKK7) (Merritt et al. J.
Biol. Chem. 274:10195-10202, 1999). Secondary testing, using an
independent set of siRNA with distinct targeting sequences,
confirmed that both kinases were the relevant targets (FIG. 8A).
Supporting the biological relevance of this finding, MKK7 and its
homolog, MKK4, are the canonical activators of JNK1-3 (Tournier et
al. Proceedings of the National Academy of Sciences of the United
States of America 94:7337-7342, 1997), key regulators of RGC cell
death (Sun et al. Mol. Vis. 17:864-875, 2011; Ribas et al.
Neuroscience 180:64-74, 2011; Bessero et al. J. Neurochem.
113:1307-1318; Fernandes et al. Neurobiology of Disease
46(2):393-401, 2012). As an additional validation that our siRNA
result was specifically due to MAP3K12/DLK pathway inhibition, RGCs
were isolated from mice containing a floxed allele of Dlk (Miller
et al. Nat. Neurosci. 12:387-389, 2009) or wildtype controls and
then transduced with adenovirus expressing the P1 bacteriophage Cre
recombinase or a GFP control. Similar to the results with RNA
interference, genetic deletion of MAP3K12/DLK led to increased RGC
survival (FIG. 8B).
MAP3K12/DLK Downregulation Promotes Long-Term Survival and Function
of RGCs In Vitro
The kinetics of RGC cell death following MAP3K12/DLK knockdown were
studied Immunopanned RGCs were transfected with Dlk siRNA, or a
nontargeting control, and followed over time. By 24 hours, Dlk
siRNA efficiently reduced MAP3K12/DLK expression at both the mRNA
and protein levels. Consistent with MAP3K12/DLK being a major
activator of JNK in injured RGCs, MAP3K12/DLK knockdown inhibited
JNK phosphorylation, indicating attenuation of downstream JNK
signaling (FIG. 6C). By 48 hours there was a clear survival effect
(FIG. 6D). While there were very few live control cells, RGCs
transfected with Dlk siRNA had greater than 50% viability. The
prosurvival effect of MAP3K12/DLK inhibition persisted for at least
3 weeks. Dlk mRNA levels stayed low throughout this period,
consistent with reports that siRNA knockdown in post-mitotic
neurons can be long-lived (Tanaka et al. Neurochem. Res.
36:1482-1489, 2011; Tanaka et al. J. Neurosci. Methods 178:80-86,
2009).
Axonal injury typically reduces the expression of many RGC specific
markers secondary to the downregulation of the Brn3 family of
transcription factors (Yang et al. Investigative Opthamology &
Visual Science 48:5539-5548, 2007). However, in RGCs with
MAP3K12/DLK knockdown, Brn3 continued to be expressed (FIG. 6E).
This showed that MAP3K12/DLK may be a relatively upstream injury
signal and that injured RGCs, in the absence of MAP3K12/DLK
signaling, maintain characteristics of uninjured RGCs. At the
functional level, patch-clamp recordings showed that RGCs kept
alive for two weeks with Dlk siRNA continue to generate action
potentials in response to depolarizing current (FIG. 6F).
MAP3K12/DLK Inhibition Promotes RGC Survival In Vivo Following
Optic Nerve Injury
To test the role of MAP3K12/DLK on RGC survival in vivo, the mouse
optic nerve crush model was used. In response to axonal injury,
50-75% of RGCs die by two weeks (Li et al. Investigative
Ophthalmology & Visual Science 48:5539-5548, 2007). Mice
carrying a floxed allele of Dlk (Dlkfl/f1) were injected
intravitreally with a self-complementary, capsid-modified
adenoassociated virus 2 (AAV2)(Petrs-Silva et al. Mol. Ther.
17:463-471, 2009; Petrs-Silva et al. Mol. Ther. 19:293-301)
expressing Cre. Injection of the AAV2-Cre resulted in Cre
expression in nearly 100% of RGCs (FIG. 9A). One week after
injection, to allow sufficient time for Cre-mediated deletion of
Dlk (FIG. 9B), optic nerve crush was performed. Ten days later,
retinal flatmounts were prepared and stained for the RGC-specific
marker .beta.III-tubulin and the number of surviving RGCs was
determined. Compared to control animals (Dlk+/+ mice injected with
AAV2-Cre or Dlkfl/fl mice in the absence of Cre), Dlkfl/fl mice
injected with AAV2-Cre showed a 75% reduction in RGC loss (FIG.
9C). This increase in RGC survival was associated with decreased
JNK phosphorylation (FIG. 9D) and c-Jun expression (FIG. 9E),
markers of JNK signaling (Derijard et al. Cell 76:1025-1037, 1994;
Angel et al. Cell 55:875-885, 1988). These results show that
MAP3K12/DLK may be the primary kinase responsible for JNK pathway
activation following axonal injury.
Axonal Injury Upregulates MAP3K12/DLK Expression Through a
Posttranscriptional Mechanism
The mechanism of MAP3K12/DLK regulation was examined next.
Surprisingly, and unlike other members of the JNK cascade,
MAP3K12/DLK protein is undetectable in uninjured RGCs both in vitro
and in vivo (FIG. 10A, FIG. 10B-left panel). However, culturing
RGCs in vitro (which necessarily involves axotomy and cell injury)
and transection in vivo both lead to robust upregulation of
MAP3K12/DLK protein (FIG. 10A, FIG. 10B-right panel). In vitro,
MAP3K12/DLK protein levels increased more than 10-fold within 18
hours from the initiation of cell culture. In contrast, Dlk
transcript levels remained relatively constant during this period
(FIG. 10A), indicating that increased translation and/or decreased
protein turnover must underlie the mechanism of MAP3K12/DLK
upregulation. In D. melanogaster, Wallenda/DLK is
post-translationally regulated by the E3 ubiquitin ligase Highwire
(Collins et al. Neuron 51:57-69, 2006; Xiong et al. J. Cell Biol.
191:211-223, 2010). However, mice with a brain-specific conditional
knockout of Phr 1 (the vertebrate Highwire homolog) show no
difference in the overall brain levels of MAP3K12/DLK protein
(Bloom et al. Genes Dev. 21:2593-2606, 2007). Furthermore,
knockdown of PHR1 in RGC cultures did not affect MAP3K12/DLK
levels, showing that either PHR1 regulates MAP3K12/DLK levels only
in certain settings/neuronal subtypes or that MAP3K12/DLK levels in
vertebrates are regulated by another as yet unidentified
protein.
Since MAP3K12/DLK downregulation promotes RGC survival, the
complementary hypothesis that increased MAP3K12/DLK expression can
trigger RGC cell death was tested. Adenovirus was used to
overexpress GFP, MAP3K12/DLK or a kinase-dead (KD) version of
MAP3K12/DLK (K185R) (Robitaille et al. Cell Death Differ.
11:542-549, 2004). Primary RGCs were transduced and survival
measured 48 hours later. Consistent with the present model,
wildtype MAP3K12/DLK overexpression hastened cell death, while
overexpression of K185R MAP3K12/DLK, which functioned as a
dominant-negative mutant as assessed by JNK phosphorylation,
actually promoted RGC survival (FIG. 10C).
Discussion
Large-scale RNAi-based phenotypic screens in lower organisms have
successfully identified genes involved in the rescue of neuronal
degeneration (Bhattacharya et al. J. Neurosci. 32:5054-5061, 2012;
Dimitriadi et al. PLoS Genet. 6:e1001172, 2010; Schulte et al. PLoS
ONE 6:e23841, 2011). Parallel screens utilizing primary vertebrate
neurons, however, have been more difficult due to the challenges
working with and transfecting primary neuronal cell cultures. Using
a magnetic nanoparticle-based method, easily compatible with
automation, these challenges were overcome to perform the first
kinome-wide survival screen using a disease-relevant primary
neuron. This global and unbiased approach led to the identification
of MAP3K12/DLK signaling as a key cell death pathway in RGC
degeneration. Moreover, it established the proof-of-principle for a
whole-genome scan in primary RGCs to identify additional potential
neuroprotective pathways and drug targets.
Although the JNK pathway may be involved in both traumatic and
glaucomatous models of optic neuropathy (Sun et al. Mol. Vis.
17:864-875, 2011; Ribas et al. Neuroscience 180:64-74, 2011;
Bessero et al. J. Neurochem. 113:1307-1318; Fernandes et al.
Neurobiology of Disease 46(2):393-401, 2012), the mechanism by
which axonal injury leads to JNK activation in RGC cell bodies has
been unclear. The present results show that MAP3K12/DLK may be the
as-yet-unidentified trigger for JNK activation and cell death in
injured RGCs.
The present results, in addition to implicating MAP3K12/DLK in
neurodegenerative RGC cell death, also show that MAP3K12/DLK may be
important in other forms of CNS neurodegenerative cell loss. The
finding that MAP3K12/DLK is required for the death of freshly
cultured RGCs indicates that MAP3K12/DLK does more than just
retrograde axonal injury signaling because the cell preparation and
purification process completely strips the cells of detectable
axonal and dendritic processes. Upregulated MAP3K12/DLK may
contribute to the cell death process directly within the RGC's cell
body.
The JNK signal transduction pathway consists of multiple branches
that feed into one or more of the JNKs (Weston et al. Curr. Opin.
Cell Biol. 19:142-149, 2007). Although a possible approach to RGC
neuroprotection is to directly block the pathway downstream with
small molecule JNK inhibitors, such non-specific inhibition of the
entire pathway may not be an ideal therapeutic strategy since JNK
signaling has a number of important physiologic roles, such as
tumor suppression (Davies & Tournier Biochem. Soc. Trans.
40:85-89, 2012). However, the present finding that the MAP3K12/DLK
branch is the major pathway leading to proapoptotic JNK activation
following RGC injury makes possible a more fine-tuned and specific
approach.
REFERENCES
All publications, patent applications, patents, and other
references mentioned in the specification are indicative of the
level of those skilled in the art to which the presently disclosed
subject matter pertains. All publications, patent applications,
patents, and other references are herein incorporated by reference
to the same extent as if each individual publication, patent
application, patent, and other reference was specifically and
individually indicated to be incorporated by reference. It will be
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Although the foregoing subject matter has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be understood by those skilled in the art
that certain changes and modifications can be practiced within the
scope of the appended claims.
* * * * *